Table 1.
Muscle biopsies used in study.
Fig 1.
Co-immunostaining of muscle sections with utrophin and fetal and developmental myosin antibodies.
(A-C) No sarcolemmal utrophin staining in control (C3), weak labelling in BMD (P7) and strong labelling in DMD (P15) muscle sections used for intensity analysis. (D-F) Utrophin merged with fetal and developmental (develop.) myosin staining to identify regenerating fibres, which were excluded from the analysis in this experiment. White bar—50μM.
Fig 2.
Quantification of utrophin in mature fibres by Metamorph software following normalisation to spectrin.
(A) Utrophin intensity (arbitrary units; A.U.) quantified in control (blue dots), carrier (manifesting P1 and P2; non-manifesting P3 and P4; grey dots), BMD (P5-7; green dots), BMD/DMD intermediate (P8; green dots) and DMD (P9-15; red dots) quadriceps muscle. The dot plot depicts the median utrophin intensity (± IQR) in mature muscle fibres from replicate experiments performed on the same muscle but stained and quantified on separate occasions (80 fibres in total analysed). Each point represents a single sarcolemmal intensity reading on one individual myofibre, normalised to spectrin and expressed as a percentage of the average intensity in the 3 non-pathological controls. Intensity measurements distinguish BMD from DMD muscle biopsies. Data are from 2 replicate experiments combined and a Kruskal-Wallis one way analysis of variance followed by Dunn’s test was performed for all comparisons. (B) The variability between replicate experiments performed on control, BMD and DMD muscle biopsies. Wilcoxon matched pair analysis used for comparison between replicate experiments (* p<0.05;** p<0.01; *** p<0.001; n/s, not significant)
Table 2.
Normalised utrophin intensity (mean ± standard error of mean).
Fig 3.
Quantification of utrophin intensity on regenerating fibres in BMD and DMD.
(A). Utrophin intensity (arbitrary units; A.U.) and (B) fibre diameter quantified in both fetal and developmental (fetal/develop) myosin negative (light green/red) and positive (dark green/red) muscle fibres in 3 BMD (P5, 6 and 7) and 6 DMD (P9, P10, P11, P12, P14 and P15) (pooled analysis). A single sarcolemmal utrophin measurement was taken from randomly selected fetal and developmental myosin positive and negative fibres from 4 fields of view in areas of muscle regeneration. (n = 39) for P3, (n = 27) for P7, (n = 46) for P8, (n = 60) for P2, (n = 45) for P3, (n = 55) for P4, (n = 53) for P17, (n = 25) for P19 and (n = 49) for P21 where n = total number of fetal/developmental myosin positive and negative fibres analysed for each muscle biopsy. A Kruskal-Wallis one way analysis of variance followed by Dunn’s test was performed (median and IQR depicted; * p<0.05; *** p<0.001, n/s, not significant).
Fig 4.
Correlation of utrophin immunostaining with β-dystroglycan and ɣ-sarcoglycan in muscle fibres.
(A, B) ɣ-sarcolgycan intensity (arbitrary units; A.U.) on muscle fibres from 4 (P11) and 8 (P12) year old DMD muscle and (C, D) β-dystrogylcan intensity (A.U.) in muscle fibres from 4 (P11) and 8 (P12) year old DMD patients revealed significant correlations with utrophin using Pearson’s and Spearman’s test. Regression line with 95% confidence intervals is shown. (E) The intensity of β-dystroglycan was significantly reduced in DMD P11 and 12 following normalisation to spectrin and expression relative to control muscle biopsy, C2. (One Way ANOVA with Tukey’s: ** p<0.01; *** p<0.001)
Fig 5.
Quantification of regeneration in BMD and DMD.
(A) Regenerating myofibres (i.e. fetal and developmental myosin positive), quantified in 3 BMD (green) and 6 DMD (red) muscle biopsies show variable regeneration levels in BMD. Counts obtained from 10 random fields (except P7 and P9 where 9 fields from each were quantified) and expressed as a percentage of the total number of fibres. (B) Pooled analysis of total regenerating fibres showed double the number in DMD compared to BMD and (C) increased mean intensity of both myosins was seen in DMD ((n = 100), developmental; (n = 124), fetal total fibres) compared to BMD ((n = 18), developmental; (n = 27), fetal total fibres analysed) muscle fibres when sections were stained with either fetal or developmental myosin only. No regenerating fibres were present in BMD muscle section P5. Kruskal-Wallis followed by Dunn’s test performed (median and IQR depicted; * p<0.05; ** p<0.01; *** p<0.001, n/s—not significant).
Fig 6.
Correlation of the percentage of regenerating fibres with functional motor score in BMD and DMD.
Correlation between functional motor score (assessment from a total score of 40) and the level of regeneration in quadriceps muscle from BMD (n = 2) and DMD (n = 6) patients. Dashed lines show 95% confidence intervals.