Article Figures & Data
Figures
- Figure 1. Disruption of RhoA-actin-serum response factor (SRF) pathway in dystrophic cells and patient muscle biopsies.
To mimic dystrophic conditions, H2K 2B4 myotubes were transfected with siRNAs targeting the Dmd gene (Dmd siRNA, red), and a non-targeting (NT siRNA, black) siRNA was used for control. (A, B) Dystrophin was successfully down-regulated at (A) protein and (B) the transcript level. (C, D, E) In addition, the absence of dystrophin resulted in (C) altered RhoA activity, (D) reduced F/G-actin levels, and (E) greater cytoplasmic MRTF accumulation (loading controls: nuclear TBP, and cytoplasmic β-actin). For gene transcript studies, cells were placed is serum free media and collected over a 24-h time course (double plotted-48 h-to show oscillation pattern). (F) Circadian time (CT) gene expression data indicates diurnal oscillation patterns and down-regulation of pertinent core clock genes (Per1 and Per2) and down-stream targets for RhoA-actin-SRF (Nr1d1 and Acta1; housekeeping gene—Gapdh). (G) Muscle biopsies were obtained from an array of patients with different mutations or deletions in the dystrophin genes, and indicate down-regulation of RhoA-actin-SRF target genes in most cases (housekeeping genes—RPL13a). All samples were normalised to a pooled skeletal muscle sample from two healthy volunteers, as indicated by red line. For RhoA GLISA, F/G actin ratio and MRTF Westerns, data were normalised to NT control (n = 3; two-tailed t test). For RT-qPCR CT data, the nadir was determined as the minimum value across both treatments (Dmd and NT siRNAs) and CTs and applied to all samples; nadir normalised to 1 (n = 2–3 for each siRNA and time point; two-way ANOVA with Bonferroni post hoc test performed). Mean values reported with SEM; ***P < 0.001, **P < 0.01, *P < 0.05. For uncropped Western blot images and loading controls, see Source Data Fig 1.
Source data are available for this figure.
Source Data for Figure 1[LSA-2021-01014_SdataF1.tif]
- Figure 2. Diurnal changes in RhoA-MRTF-serum response factor (SRF) cascade components in tibialis anterior of dystrophin-utrophin (dKO) mouse model.
(A) Tissues were collected over a 24-h time course and double plotted (48 h) to better illustrate the (A) oscillation pattern of core clock genes in tibialis anterior (TA) of 5-wk-old dKO animals which were significantly altered compared to C57BL10 (Zeitgeber- ZT). Down-stream targets for RhoA-actin-SRF pathway (Nr1d1, Rora1, and Acta) and indeed Srf were also altered (Gapdh used as housekeeping gene). (B, C) F/G-actin protein ratios in dKO mice were significantly down-regulated, (C) as were cytoplasmic MRTF fraction levels (loading control: nuclear TBP, and cytoplasmic vinculin). In addition, C57BL10 animals exhibit diurnal changes in F/G actin ratio and MRTF fraction levels. Light and dark periods represented by outlined (light) and solid bars (dark). For RT-qPCR ZT data, the nadir was determined as the minimum value across both genotypes and ZTs and applied to all samples; nadir normalised to 1 (n = 3–4 for each for each genotype and time-point). For F/G actin ratio and MRTF westerns, data were normalised to C57BL10 (n = 3–4). One or two-way ANOVA with Bonferroni post hoc test performed. Mean values reported with SEM; ***P < 0.001, **P < 0.01, *P < 0.05. For uncropped Western blot images and loading controls, see Source Data Fig 2.
Source data are available for this figure.
Source Data for Figure 2[LSA-2021-01014_SdataF2.tif]
- Figure S1. F/G actin ratios in C57BL10, and mdx and dKO littermates.
F/G-actin protein ratios from the tibialis anterior (TA) of 5-wk-old dKO were significantly lower than littermate mdx and C57BL10 animals (ZT1). N = 3–4 for each for each genotype; one-way ANOVA with Bonferroni post hoc test performed. Mean values reported with SEM; ***P < 0.001, **P < 0.01, *P < 0.05.
- Figure 3. Perturbed circadian rest-activity and response to jet-lag in dystrophic mouse model.
(A) Dystrophin Western blot shows the absence of protein in the SCN of mdx animals (vinculin used as loading control). (B) Locomotor behaviour of mdx and C57BL10 mice were assessed and representative wheel-running activity blots (B) shown; 12 h:12 h light:dark cycle (LD), dark:dark (DD), and light:light (LL). (C, D, E, F, G, H) Data analysed and represented as graphs: (C) behaviour during LD, (D) after 6-h phase advancements, (E) phase angle on release into DD, (F) behaviour during DD, (G) phase delay after a light pulse 4-h post exercise commencement, (H) and behaviour during LL. (I) Weights of mice undergoing 5-wk jetlag protocol assessed (C57BL10, mdx age matched and mdx weight matched), as well as total body weight of jetlagged (JL) and non-jetlagged (NJL) mdx and C57BL10 cohorts. t test for comparisons between two groups (two-tailed). For weights recorded over the 5 wk of jetlag: black asterisk on right-one-way ANOVA comparing all three cohorts; dollar sign-one way ANOVA with Bonferroni post hoc between each cohort at each time point. For body weights with JL and NJL, one-way ANOVA with Bonferroni post hoc test performed (For wheel running activity n = 5–6, jetlag study n = 3–4; ***P < 0.001, **P < 0.01, *P < 0.05). For uncropped dystrophin Western blot and loading control, see Source Data Fig 3.
Source data are available for this figure.
Source Data for Figure 3[LSA-2021-01014_SdataF3.tif]
- Figure 4. Altered expression of clock genes in tibialis anterior of mdx mouse model.
Tissues were collected over a 24-h time course and double plotted (48 h) to better illustrate the oscillation pattern of core clock genes in tibialis anterior (TA) of 20 wk old mdx animals which were significantly altered compared with C57BL10 (Zeitgeber- ZT). Down-stream targets for RhoA-actin-serum response factor pathway (Nr1d1, Acta, and Rora1) were also altered (Atp5b used as housekeeping gene). Light and dark periods represented by outlined (light) and solid bars (dark). The nadir was determined as the minimum value across both genotypes and ZTs and applied to all samples; nadir normalised to one (n = 3–4 for each for each genotype and time-point; two-way ANOVA with Bonferroni post hoc test performed). Mean values reported with SEM; ***P < 0.001, **P < 0.01, *P < 0.05.
- Figure S2. Utrophin protein and transcript levels altered in dystrophic models.
(A, B) Transcript levels (left), Western blot quantification (middle), and representative Western blots (right; total protein quantified for normalisation) for utrophin shown for (A) mdx and C57BL10 mice and (B) H2K 2B4 myotubes. (C) UTRN gene expression down-regulated in all but one Duchenne muscular dystrophy muscle biopsy sample. (D) Linear regression plotting PER1, PER2, and NR1D1 versus UTRN gene expression indicates significant correlation pattern with PER1 (R = 0.1687) and PER2 (R = 0.4542). Data normalised to NT siRNA or C57BL10; t test for comparisons between two groups (two-tailed; ***P < 0.001, **P < 0.01, *P < 0.05). N = 3–4. For uncropped Western blot images and loading controls, see Source Data Fig S2.
Source data are available for this figure.
Source Data for Figure S2[LSA-2021-01014_SdataFS2.tif]
- Figure S3. In vitro dose response curves for Srf and Acta.
H2K 2B4 myotubes were transfected with 3 nM, 30 nM and 100 nM of siRNA targeting the Srf, Acta and Dmd genes (Srf siRNA, blue; and Acta siRNA, grey), and a non-targeting (NT siRNA, black) siRNA was used for control. Gene expression data indicates significant and sustained knock-down of respective genes at low doses (housekeeping gene— Gapdh). Data normalised to NT siRNA; n = 3; two-way ANOVA with Bonferroni post hoc test performed. Mean values reported with SEM; ***P < 0.001, **P < 0.01, *P < 0.05.
- Figure 5. Abrogation of alternative RhoA-actin-serum response factor (SRF) components leads to a reduction in the expression of SRF target genes.
H2K 2B4 myotubes were transfected with 100 nM siRNAs targeting the Srf, Acta and Dmd genes (Srf siRNA, blue; Acta siRNA, grey; and Dmd siRNA, red), and a non-targeting (NT siRNA, black) siRNA was used for control. Gene expression data indicate successful knock-down of respective genes (Srf, Acta and Dmd) and down-regulation of pertinent core clock genes (Per1 and Per2) and other down-stream targets for RhoA-actin-SRF (Nr1d1 and Acta1; housekeeping gene—Gapdh). Data normalised to NT siRNA; n = 3; two-way ANOVA with Bonferroni post hoc test performed. Mean values reported with SEM; ***P < 0.001, **P < 0.01, *P < 0.05.
- Figure 6. Schematic illustrating RhoA-actin-serum response factor (SRF) signalling pathway.
In healthy muscle, systemic cues activate RhoA in peripheral tissues which in turn regulates diurnal polymerisation (F-actin) and de-polymerisation (globular (G)-actin) of actin. During the de-polymerisation stage, G-actin preferentially binds to myocardin-related transcription factor (MRTF). However, when this shifts to the polymerisation phase, the G-actin pool diminishes and unbound MRTF translocates into the nucleus and influences expression of SRF. SRF activation regulates transcription of SRF target genes including the core clock genes—Per1 and Per2, as well as secondary loop clock genes—Rora1 and Nr1d1, and cytoskeletal genes such as Acta. When dystrophin is absent, F-actin is not stable and the cell shifts to the de-polymerised state. G-actin binds to MRTF and therefore SRF transcription is hampered.
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