Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis

Figures

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  Figure 1. Cross section of gluteal muscle from an RER-susceptible STD. (A) Hematoxylin and Eosin stain showing numerous mature myofibers with internalized myonuclei 20×. (B) ATPase stain with pH 4.4 acid preincubation showing three muscle fiber types.

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  Figure 2. Transcriptomic and proteomic differential expression in RER-susceptible Standardbreds. (A). Volcano plot depicting the probability of observing the estimated change in gene expression (−log10 scale) on the Y axis and the degree of fold change differences (log2 scale) on the X axis. (B). Volcano plot depicting estimated p-values from differential expression of protein analysis versus log2 fold change. There were 791 differentially expressed genes (green dots; FDR ≤ 0.05) and 62 differentially expressed proteins (blue dots; FDR ≤ 0.10) between RER-susceptible and control horses. The genes/proteins with the highest and lowest fold changes are annotated on each graph.

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  Figure 3. Illustration of convergent pathways for differentially expressed genes and proteins. The number and cellular location of up and downregulated differentially expressed genes (DEG) categorized by convergent pathways for GO terms in biological processes (see Table S4 for specific GO terms and genes) were combined with the number of up and down regulated significantly differentially expressed proteins (DEP) (see Table 3 and Table 4 for specific proteins) in RER-susceptible compared to control horses. The number of genes corresponds to the number of genes in the GO term with the largest gene set for the convergent pathway (Table S4). DEP and DEG for processes of purine nucleotide metabolism, the electron transfer system (ETS), tricarboxylic acid cycle (TCA) and fat metabolism are depicted within the mitochondrion. Between episodes of rhabdomyolysis, RER-susceptible horses showed upregulation of cellular inflammation, cellular/oxidative stress response, cell regeneration, protein modification and sarcomere proteins. Metabolic pathways were highlighted in the GO analysis in RER-susceptible horses with a notable increase in expression of proteins and decrease in expression of genes…

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  Figure 4. Depiction of the differentially expressed downregulated (red) and upregulated (green) genes that impact sarcoplasmic reticulum calcium stores (CASQ1, STIM1) and myoplasmic stores (ANK2) or modulate calcium release by the calcium release channel RYR1. The dihydropyridine receptor (CANA2D1) activates RYR1 resulting in sarcoplasmic reticulum calcium release. Down-regulated Protein NipSnap homolog 2 (GBAS) acts as a positive regulator and upregulated Ras related glycolysis inhibitor and calcium channel regulator (RRAD) an inhibitor of CANA2D1. Junctophilin-2 (JP2) inhibits RYR1 calcium release. S100 calcium binding protein A11 (S100A11) colocalizes with S100A1 which increases RYR1 calcium release. Calsequestrin-1 (CASQ1) is a high-capacity, moderate affinity, calcium-binding protein that regulates the release of luminal calcium via RYR1. Calcium/calmodulin-dependent protein kinase type II subunit alpha (CAMK2) and protein kinase A (PKA) phosphorylate RYR1, which increases RYR1 calcium release. Beta adrenergic (epinephrine) stimulation activates protein kinase A (PKA) via an increase in adenylyl cyclase which is opposed by phosphodiesterase (PDE4B). Stromal interaction molecule 1…

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  Figure 5. Enrichment map for GO biological processes. Up-regulated differentially expressed genes were enriched for biological processes including inflammation/immune response and reactive oxygen species/antioxidant responses. The size of the vertex indicates the number of DE target genes enriched for that term. The color of the vertex indicates the adjusted p-value and the edges (lines) connecting the vertices reflect DE target genes that were common between the GO terms.

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  Figure 6. Pathway enrichment for DEG and DEP. (A). Enriched Reactome pathways from the amalgamated analysis of B. down-regulated DEP and DEG and (B). Up-regulated DEP and DEG.

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  Figure 7. The concentrations of ROS, glutathione and CoenzymeQ10 in RER-susceptible compared to control horses. (A). ROS concentrations (B). Glutathione concentrations. (C). CoQ10 concentrations. Only CoQ10 concentrations were significantly higher (p = 0.0072) in RER-susceptible horses. ** indicates p < 0.01.

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  Figure 8. The differentially expressed proteins (dark green upregulated and dark red downregulated) and genes (light green italics upregulated, light red italics downregulated) involving antioxidants, the tricarboxylic acid cycle (TCA), cysteine synthesis, and glutathione synthesis in muscle from RER-susceptible compared to control horses. RER-susceptible horses had increased expression of complexes I and III in the electron transfer system which generated reactive oxygen species (ROS). RER-susceptible horses had increased expression of superoxide dismutase (SOD) which produces H2O2 and decreased expression of antioxidant proteins catalase (CAT) and the cysteine-based antioxidant peroxiredoxin 2 (PRDX2) as well as increased expression of the cysteine-based genes sulfiredoxin (SRXN1 7.98 log2FC), thioredoxin, and thioredoxin reductase, which all reduce H2O2 to H2O. Genes involved in the transport and synthesis of cysteine from methionine and the transport and synthesis of the antioxidant glutathione from cysteine, glycine and glutamate were DEG in RER-susceptible versus controls. Proteins (increased) and genes (decreased) in the TCA cycle that produce the glutamate precursor alpha…