Table 1.
The number of controls, RER-susceptible (no dantrolene) and RER mares treated with dantrolene (RER-D).
Fig 1.
Depiction of the cellular location of differentially expressed proteins and select differentially expressed genes in RER-susceptible horses.
Increased expression is in green and decreased expression in red. Proteins that also had differential expression of their encoding gene are underlined. Differentially expressed genes (italics) relating to Ca2+regulation are shown in orange. Ca2+ is depicted by purple circles. Full names of genes and proteins are found in Tables 2–4. RER-susceptible horses between episodes of rhabdomyolysis had increased expression of ANK1, which links sarcoplasmic reticulum to myofilaments, Ca2+ binding proteins within the sarcoplasmic reticulum (CASQ1, SRL, HRC), the Ca2+ release channels ITP3R and RYR1, as well as regulators of RYR1 [calmodulin (CALM2), calsequestrin (CASQ1), PKD2, and CACHD1 (via DHPR)]. Proteins involved in oxidative stress (PRDX1, PRDX2, SELENBP1) and protein degradation [Ca2+-activated calpain (CAPN3), ubiquitination (UBC, UBE2V2), proteasome (PSMA4)] were differentially expressed. Decreased expression was found for Ca2+ activated proteins [SERCA2 (ATP2A2), troponins (TNN1,C,T) myosin regulatory light chains (MYL2,3)] as well as other sarcomere proteins (ACTN3, MYOZ, FLNC, MYH1). Heat shock proteins (HSPB1, HSPA9) and protein chaperones had decreased expression in RER. Decreased expression was found for mitochondrial proteins myoglobin (MB), 6 subunits of complex I, 3 subunits of complex III, 4 subunits of complex IV and 4 subunits of complex V. ANXA2, a Ca2+ activated membrane repair protein was also downregulated and sarcolemmal structural proteins dystrophin (DMD) and syntrophin (SNTB1) were upregulated. The sodium/potassium exchanger (ATP1A2) and the sodium/ Ca2+ exchanger ATP2B4, were also differentially expressed.
Fig 2.
A potential scenario for the development of rhabdomyolysis in RER-susceptible horses derived from differentially expressed proteins and genes.
Green indicates increased and red decreased differential expression. Genes are italicized. A stressful environment is proposed to induce hyper-phosphorylation of RYR1 through beta adrenergic (epinephrine mediated) activation of calmodulin kinase II (CAMKII) which is stimulated by the DEP calmodulin (CALM2). This and other potential post-translational modifications of RYR1 (oxidation/nitrosylation) allow excessive Ca2+ (purple circles) release of high sarcoplasmic reticulum Ca2+ stores in RER-susceptible horses through RYR1 (orange) when RYR1 opening is stimulated during exercise by the voltage gaited Ca2+ channel in the t-tubule (turquoise). Myoplasmic Ca2+ interacts with troponin I (TNNI) and, when not adequately pumped back into the sarcoplasmic reticulum (ATP2A2) or out of the cell (ATP2B4), Ca2+ produces persistent contracture of the sarcomere (pink and purple). Mitochondria buffer myoplasmic Ca2+ through VDAC uptake where Ca2+ stimulates ATP production. However, in excess, Ca2+ uncouples (UCP2) oxygen consumption from electron transport and ATP production and releases reactive oxygen species (ROS). Antioxidants such as peroxiredoxin (PRDX1, PRDX2) counteract ROS to prevent oxidative stress. Excessive mitochondrial matrix Ca2+ results in formation of the membrane transition pore (BAX, SLC25A6, ATP5), loss of membrane potential, release of cytochrome C and apoptosis. Calcium activation of proteases such as calpain (CAPN3) results in degradation of myofilaments, cellular proteins and cell membranes resulting in the release of muscle proteins such as creatine kinase (CK) into the blood stream. Calcium activated ANAX2 participates in repair of membranes that have increased dystrophin (DMD) and syntropin (SNTB1) content. Degraded proteins are ubiquitinated (UBC, UBE2B2) and processed in the proteasome (PSMA4).
Table 2.
Differentially expressed muscle proteins (DEP) with increased expression comparing horses susceptible to recurrent exertional rhabdomyolysis with control.
Table 3.
Differentially expressed mitochondrial proteins (DEP) with decreased expression comparing horses susceptible to recurrent exertional rhabdomyolysis with control.
Table 4.
Differentially expressed nonmitochondrial proteins (DEP) with decreased expression comparing horses susceptible to recurrent exertional rhabdomyolysis with control.
Fig 3.
Depiction of the location of differentially expressed proteins and genes within the mitochondrial tricarboxylic acid cycle, electron transport system and mitochondrial Ca2+regulation.
Increased expression is in green for protein and yellow for genes. Decreased expression is in orange for proteins and pink for genes. Note the strong impact of RER-susceptibility on the electron transfer system and channels related to Ca2+ uptake (VDAC) or overload (mitochondrial permeability transition pore).
Fig 4.
(A) GO and KEGG pathways enriched for DEG and DEP. The size of the dots represents the ratio of DEG or DEP to the background used in the enrichment analysis. Since the background of DEG differs from that used for DEP, the points are color coded to note the number of DEG or DEP in each pathway. (B) Heatmap of the information content (IC) shared between two pathways. The asterisk (*) denotes two pathways that share an IC > 0.65.
Fig 5.
Heatmap illustrating the individual DEG and DEP found within enriched GO and KEGG pathways.
Darker colors indicate lower log2 fold change difference in gene or protein expression in RER-susceptible compared to control horses.
Fig 6.
Principal component (PC) analysis of transcriptomic data for control, RER-susceptible (RER) and RER horses treated with dantrolene (RER-D).
(A) PC1 versus PC2. Note that RER-susceptible horses cluster separately from overlapping RER-D and controls. (B) PC1 and PC3. The proportion of variance explained by the RER and control groups is reduced by PC3. (C) MA plot depicting the average expression of gene transcripts compared to the log2 fold change of differentially expressed genes comparing RER-susceptible to controls. There were 812 differentially expressed genes (red dots) between RER-susceptible and control horses. (D) MA plot showing the average expression of gene transcripts compared to the log2 fold difference in differentially expressed genes in RER-D compared to controls. There were no significant differentially expressed genes observed between RER-D and controls.