Diaphragm-specific effects of L-citrulline in mdx mice highlight its potential as adjuvant of standard therapy in Duchenne muscular dystrophy

Authors: TULIMIERO, LISAMAURA - University Of Bari; BOCCANEGRA, BRIGIDA - University Of Bari; MANTUANO, PAOLA - University Of Bari; MELE, ANTONIETTA - University Of Bari; BELLIS, MICHELA - University Of Bari; LENTI, ROBERTA - University Of Bari; SANARICA, FRANCESCA - University Of Bari; CIRMI, SANTA - University Of Bari; CONTE, ELENA - University Of Bari; CAPPELLARI, ORNELLA - University Of Bari; SHERRARD, AMBER - Children'S Nutrition Research Center (CNRC); GREEN, ARDAWNA - Children'S Nutrition Research Center (CNRC); SRINVASA, MIRA - Children'S Nutrition Research Center (CNRC); FIOROTTO, MARTA - Children'S Nutrition Research Center (CNRC); LUCA, ANNAMARIA - University Of Bari

Submitted to: British Journal of Pharmacology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/22/2026
Publication Date: 4/22/2026
Citation: Tulimiero, L., Boccanegra, B., Mantuano, P., Mele, A., De Bellis, M., Lenti, R., Sanarica, F., Cirmi, S., Conte, E., Cappellari, O., Sherrard, A.D., Green, A., Srinvasa, M., Fiorotto, M.L., De Luca, A. 2026. Diaphragm-specific effects of L-citrulline in mdx mice highlight its potential as adjuvant of standard therapy in Duchenne muscular dystrophy. British Journal of Pharmacology. https://doi.org/10.1111/bph.70437.
DOI: https://doi.org/10.1111/bph.70437

Interpretive Summary: Duchenne muscular dystrophy is the most prevalent genetic disease in boys that causes severe progressive muscle wasting, loss of muscle function in early life, loss of mobility, and early death. There is no cure, but some treatments that can slow down the loss of function. A decrease of nitrous oxide (NO) production in muscle is one of the consequences of the disease and contributes to its pathology. L-citrulline, a non-essential amino acid, is a precursor of the amino acid arginine from which NO is made. The concentrations of both arginine and citrulline in dystrophic muscle are low in the dystrophic muscle. In this study L-citrulline was supplemented from a young age in the diet of mdx mice for up to 9 weeks. The mdx mouse is a model that develops many of the same the same defect as the human form of the disease. We found that supplementation with citrulline increased both its concentration and that of arginine in the muscle and plasma. It improved arm muscle strength and improved the movement and contraction of the diaphragm, the muscle that is most affected in this disease. Genes associated with calcium handling in the diaphragm and that regulate muscle contraction were also improved. The study suggests that citrulline (found in significant quantities in watermelon) may be a useful dietary supplements for boys who have this disease.

Technical Abstract: Background and purpose: The absence of the protein dystrophin in Duchenne muscular dystrophy (DMD) leads to progressive muscle weakness, failing regeneration and deregulation of nitric oxide (NO) signalling. We focused on L-citrulline, a precursor of L-arginine, required for NO production in muscle, which is reduced in dystrophic mdx muscle. Experimental approach: L-Citrulline was administered (2 mg g-1 die-1), through diet, in comparison and/or in combination with prednisolone (1 mg kg-1, 5 days per week subcutaneously) to 4- to 5-week-old mdx mice for 8 weeks. Key results: L-Citrulline increased the levels of L-arginine, L-citrulline and L-ornithine in plasma and quadriceps of mdx mice. L-citrulline, alone or plus prednisolone, significantly improved maximal forelimb force in vivo, while ameliorating diaphragm movement amplitude and reducing diaphragm echodensity. In parallel, ex vivo, we detected a significant improvement of diaphragm force and contraction kinetics in mice treated with L-citrulline alone or in combination with prednisolone. L-citrulline also restored the expression of genes involved in Ca2+ handling during contraction (RyR1, RyR3 and SERCA), while reducing the markers of inflammation and fibrosis (CD68 and TGFß1) and ameliorating mitochondrial biogenesis-associated genes (PGC1-a and MEF2C). No effect was observed on S-nitrosylation levels of HDAC2 and on diaphragm and gastrocnemius nNOS gene expression, suggesting a NO-independent mechanism underlying the positive outcome observed. Conclusions: Our results revealed the ability of L-citrulline supplementation to ameliorate in vivo and ex vivo function of diaphragm muscle, highlighting novel metabolic and calcium-related mechanisms of potential clinical interest.