Small‐Molecule Targeting MuRF1 Protects Against Denervation‐Induced Diaphragmatic Dysfunction: Underlying Molecular Mechanisms

Figures

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  FIGURE 1. Transcriptomic profiling of the diaphragm following 12 h of UDD and identification of pathways responsive to MyoMed‐205. (A) Schematic illustration of the experimental design used to investigate the effect of small‐molecule targeting MuRF1 (MyoMed‐205, 50 mg/kg bw, intravenous delivery) upon early UDD pathophysiology in rats. (B) Principal component analysis for RNA‐seq of diaphragm muscle and identification of differentially expressed genes (DEGs, p adj < 0.05) across the distinct experimental conditions (n = 4 per group). Upregulated genes are represented in red, while downregulated genes are represented in blue. (C) Hierarchical clustered heat map highlights five clusters of DEGs identified in response to UDD and MyoMed‐205 compared with the control. (D) GSEA reveals the hallmark pathways responsive to UDD and the impacts of MyoMed‐205. Additionally, GSEA indicates the altered (E) molecular functions (GOMF) and (F) cellular compartments (GOCC) of the diaphragm muscle in response to UDD and MyoMed‐205 compared with controls. FDR, false discovery rate; GSEA, Gene Set Enrichment Analysis; p adj, adjusted p value; UDD, unilateral diaphragm denervation.

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  FIGURE 2. MyoMed‐205 protects against diaphragm fibre atrophy induced by 12 h of unilateral diaphragm denervation (UDD). (A) Representative photomicrographs of diaphragm muscle immunolabelled with antibodies against the myosin heavy chain isoforms, Type I (violet), Type IIa (light blue), Type IIb/x (black) and dystrophin (white) for morphometric analysis by fibre type. Scale bar = 100 μm. (B) Fibres' CSA, relative CSA percentual change compared with sham‐controls and relative percentual distribution by fibre type. Data are presented as mean and standard deviation (n = 8). One‐way ANOVA followed by Tukey's post hoc test was used for statistical comparisons among the groups. *p < 0.05. (C) Heat map of genes linked to muscle contraction (i.e., synaptic vesicle membrane and contractile fibre). (D) Heat map of genes linked to sarcomere structure and function (i.e., actin filament, I band and DCG). (F) Protein levels of calcium‐handling markers were assessed by Western blotting. Data are presented as mean and standard deviation of the fold change relative to the control group (n = 7–8). One‐way ANOVA followed by Tukey's post hoc test was used for statistical comparisons among the groups. *p <…

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  FIGURE 3. Unilateral diaphragm denervation enhances transcriptional activation of pathways associated with mitochondrial dysfunction and oxidative stress, while MyoMed‐205 upregulates the gene expression of antioxidant factors. (A) Heat map of genes involved in the control of mitochondrial biogenesis and dynamics. (B–C) Heat map of genes associated with mitochondrial structural and functional complexes. (D) Heat map of genes linked to mitochondrial energy production and metabolism. (E) Heat map of genes involved with ROS production and antioxidant response. ROS, reactive oxygen species; UDD, unilateral diaphragm denervation.

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  FIGURE 4. MyoMed‐205 enhances the activation of signalling pathways controlling ribosome biogenesis, protein synthesis and cell growth. (A) Heat map of Myc target genes. (B) Heat map of genes associated with the PI3K‐AKT–mTOR pathway. (C) Heat map of genes controlling ribosome biogenesis and translation initiation. (D) Protein levels of MAPK and PI3K‐Akt–mTOR pathways markers were assessed by Western blotting. Data are presented as mean and standard deviation of the fold change relative to the control group (n = 6–7). One‐way ANOVA followed by Tukey's post hoc test was used for statistical comparisons among the groups. *p < 0.05; **p < 0.01.

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  FIGURE 5. MyoMed‐205 enhances the transcriptional activation of heat shock chaperones and the UPR mechanism in the diaphragm under denervation‐induced cellular stress and catabolic state. (A) Heat map of genes from the IL6‐JAK‐STAT3 pathway. (B) Heat map of genes associated with the chaperone complex and UPR. (C) Heat map of genes linked to the ubiquitin‐proteasome system. (D) Protein levels of ubiquitin‐proteasome pathway markers were assessed by Western blotting. Data are presented as mean and standard deviation of the fold change relative to the control group (n = 8). One‐way ANOVA followed by Tukey's post hoc test was used for statistical comparisons among the groups. *p < 0.05; **p < 0.01. (E) Heat maps of genes from the p53 pathway. (F) Heat maps of genes associated with apoptosis. UPR, unfolded protein response.

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  FIGURE 6. MyoMed‐205 attenuates denervation‐induced intramuscular fat accumulation in the diaphragm by downregulating pro‐adipogenic genes. (A) Heat map of genes related to adipogenesis and intramuscular lipid accumulation. (B) Protein levels of adipogenesis pathway markers were assessed by Western blotting. Data are presented as mean and standard deviation of the fold change relative to the control group (n = 8). One‐way ANOVA followed by Tukey's post hoc test was used for statistical comparisons among the groups. (C) Representative photomicrographs of ORO staining of the diaphragm for assessing intramuscular lipid accumulation following 12 h of unilateral diaphragm denervation and the effects of MyoMed‐205. Scale bar = 50 μm. (D) Quantitative analysis of positive ORO muscle cells (i.e., intracellular lipid droplet area > 5% of total muscle fibre area). (E) Mean intracellular lipid droplet area of diaphragm muscle fibres < 2500 μm2 (predominantly oxidative metabolism) and fibres > 2500 μm2 (predominantly glycolytic metabolism). In total, 300 muscle cells were assessed per sample to estimate the average positive ORO cells and intracellular lipid content. Data are presented as mean and…

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  FIGURE 7. MyoMed‐205 mitigates denervation‐induced upregulation of genes involved with extracellular matrix collagen deposition and fibrosis in the diaphragm at the transcriptional level. (A) Heat map of genes involved in the formation of collagen trimmers. (B) Heat map of genes associated with the basement membrane. (C) Heat map of genes associated with collagen assembly and the extracellular matrix.