Fig 1.
Six-month ribitol treatment enhances matriglycan expression dose-dependently in P448L FKRP mutant mice.
(A) IIH6 immunohistochemistry for the detection of matriglycan. Saline treated as control. TA, Tibialis anterior. Dia, diaphragm. (B) Western blots for the detection of matriglycan with IIH6 antibody (with broad bands). Two samples for each dose are shown. The lower panels are α-actin as loading control. (C) measurement of the matriglycan bands by NIH ImageJ with C57 as 100% control. Saline, saline treated; 0.5g, 0.5g/kg; 5gX2, 5k/kg twice a day; 3gX3, 3g/Kg 3 times a day. * P<0.05 n = 3 compared to the saline control group. Scale bar = 150 μM.
Fig 2.
Dose-dependent improvement in muscle pathology of P448L mutant mice after ribitol treatment.
(A) H&E staining of skeletal muscles, Tibialis anterior (TA) and diaphragm. (B) Fiber size (μM in diameter) distribution as a percent of the total fibers. n = 5. (C) and (D) Percentage of centrally nucleated fibers in TA and diaphragm. n = 5. (E) Serum levels of creatine kinase. n = 10. * P<0.05 when compared with saline-treated control.
Fig 3.
Masson Trichrome staining for the measurement of fibrotic areas in the 6-month ribitol-treated P448L mutant mice.
(A) Microscopic image of the staining with blue color representing fibrotic areas and red color the remaining muscle fibers. (B-D) Measurement of fibrotic areas with NIH ImageJ. Y axis represents percentage of fibrotic area within the total section area (1 equals to 100% of area measured). * P<0.05 n = 5 when compared with saline-treated control.
Fig 4.
Effect of 6 month ribitol treatment on muscle functions.
Skeletal muscle functions were measured by treadmill (A-C) and grip force (D-E). Respiratory and cardiac functions were measured by plethysmography (F-G) and echocardiography (H-I). Sali, saline-treated; 0.5g, 0.5g/kg; 5gX2, 5k/kg twice a day; 3gX3, 3g/Kg 3 times a day. * p<0.05 n = 20 when compared with saline-treated P448L control.
Fig 5.
Detection of ribitol, ribitol-5-phosphate and CDP-ribitol and serum creatine kinase.
(A) Levels of ribitol, ribitol-5-phosphate (P) and CDP-ribitol in quadriceps muscles 24 hours after last dose of ribitol treatment. Saline treatment as controls. Numbers on X axis are g/kg; x2, 2 times a day; x3, 3 times a day. (B) Ribitol treatment from 10 months of age decreases levels of serum creatine kinase and ALT. *, p≤0.05 n = 3 (A) or n = 10 (B).
Fig 6.
Effect of ribitol on body weight, muscle function, fibrosis and life span of P448L mutant mice treated with ribitol starting from 10 months of age.
(A) Effect of ribitol treatment on body weight. (B) Ribitol effect on muscle function with grip force tests. (C) Masson Trichrome staining showed fibrotic area (represented by blue staining). (D) Measurement of Masson Trichrome staining of fibrotic areas. % of total area with blue staining. (E) Effect of ribitol treatment on life span of female and male mice. *, P<0.5 n = 20 for A, B; n = 5 for D; n = 10 for E. Sali, saline treatment as control. The number of weeks is calculated starting from the treatment at 10 months of age.