Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis
Abstract
:1. Introduction
2. Materials and Methods
2.1. RER-Susceptible Horses
2.2. Sample Collection
2.3. Muscle Histochemistry
2.4. Analyses of Signalment, Plasma CK, AST and Fiber Composition
2.5. Transcriptomics
2.5.1. RNA Extraction and Sequencing
2.5.2. Mapping and Assembling
2.5.3. RNA-Seq Count Normalization and Transformation
2.5.4. Differential Gene Expression Analysis
2.6. Proteomics
2.6.1. Sample Preparation and LC/MS/MS
2.6.2. Quantitative Data Analysis
2.7. Transcriptome and Proteome Co-Inertia Analysis
2.8. Enrichment and Pathway Analysis
2.9. Muscle Biochemistry
2.9.1. Reactive Oxygen Species (ROS)
2.9.2. Coenzyme Q10
2.9.3. Glutathione
2.9.4. Statistical Analyses of ROS, Glutathione and Coenzyme Q10
3. Results
3.1. Horses and Muscle Histochemistry
3.2. Transcriptomics
3.2.1. Expressed Gene Transcripts
3.2.2. Gene Ontology Pathway for RER Transcriptomic Analysis
3.3. Proteomics
3.3.1. Confident Protein Classification, Differential Expression and GO Analysis
3.3.2. Gene Ontology (GO) Pathway for RER Proteomic Analysis
3.3.3. Differentially Expressed Genes with Differentially Expressed Proteins
3.4. Reactome for Merged Differentially Expressed Genes and Proteins
3.5. Co-Inertia Analysis
3.6. Muscle Biochemistry
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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N | Age | Time | CK | AST | Type 1 | Type 2A | Type 2X | |
---|---|---|---|---|---|---|---|---|
Yrs | Hours | U/L 1 | U/L 2 | % | % | % | ||
Control | 7 | 3.9 ± 1.3 | 1.8 ± 0.6 | 257 ± 95 | 371 ± 96 | 10.8 ± 1.7 | 39.3 ± 9.4 | 49.8 ± 10.0 |
RER-susceptible | 9 | 4.8 ± 1.6 | 1.7 ± 0.7 | 433 ± 275 | 644 ± 564 | 16.9 ± 4.4 | 45.3 ± 13.9 | 37.4 ± 13.9 |
p value | 0.22 | 0.74 | 0.14 | 0.17 | 0.007 | 0.37 | 0.08 |
Category for GO Term | Total Number Genes | Number of GO Terms | Genes/GO Grouped Category |
Upregulated DEG | 385 | ||
Inflammation/Immune response | 62 | 5–49 | |
Cell proliferation | 31 | 5–22 | |
Cell/oxidative stress | 31 | 7–30 | |
Protein modification | 7 | 13–28 | |
Extracellular matrix | 7 | 6–26 | |
Regulatory processes | 6 | 4–23 | |
Amino acid/amine metabolism | 5 | 13–20 | |
Mitochondrial membranes | 3 | 5–10 | |
Ketone metabolism | 2 | 14–16 | |
Response to stimulus | 2 | 19–28 | |
Downregulated DEG | 305 | ||
Calcium ion regulation (CACNA2D1/CASQ) | 39 | 4–13 | |
Purine nucleotide metabolism | 32 | 5–42 | |
Electron transport | 29 | 4–54 | |
Other metabolism | 23 | 3–75 | |
Amino acid metabolism | 15 | 3–20 | |
Fat metabolism | 10 | 4–20 | |
Carbohydrate metabolism | 10 | 5–28 | |
Tricarboxylic acid cycle | 9 | 4–19 | |
Polysaccharide metabolism | 9 | 5–28 | |
Thermogenesis | 5 | 8–13 | |
Hormone secretion | 5 | 9–16 | |
Other Ion transport | 4 | 4–28 | |
Thioesters | 3 | 9–20 | |
Signaling | 3 | 3–15 | |
Actin | 1 | 5 | |
Reactome All DEG and DEP | Total Number Genes | Number of RH Terms | Genes/RH Grouped Category |
498 | |||
Cell proliferation | 45 | 11–18 | |
Immune response | 11 | 15–40 | |
NFkB Immune response | 7 | 14–17 | |
Cell/oxidative stress | 8 | 10–43 | |
Protein processing | 8 | 7–20 | |
Transcription | 7 | 12–19 | |
Metabolism | 9 | 5–47 | |
Electron transport | 7 | 11–48 | |
TCA cycle | 5 | 5–69 | |
Extracellular matrix | 5 | 5–13 | |
Signaling | 4 | 4–14 | |
Transporters | 3 | 12–14 | |
Muscle contraction | 2 | 9–19 |
Gene ID | Protein Name | Log2FC | Adj. P |
---|---|---|---|
Mitochondria | |||
Complex I | |||
NDUFB7 | Cluster of NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 7 | 1.23 | 4.75 × 10−2 |
NDUFA2 | Cluster of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2 | 0.71 | 7.20 × 10−2 |
NDUFS2 * | NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 | 0.47 | 8.84 × 10−2 |
Complex III | |||
UQCRB | cytochrome b-c1 complex subunit 7 | 1.55 | 5.20 × 10−2 |
LOC111767815 | cytochrome b-c1 complex subunit 8 | 1.35 | 4.56 × 10−2 |
LOC100053634 | cytochrome b-c1 complex subunit Rieske | 0.54 | 6.19 × 10−2 |
UQCRC1 | Cluster of cytochrome b-c1 complex subunit 1 | 0.64 | 6.19 × 10−2 |
LOC100051878 | Cluster of cytochrome b-c1 complex subunit 6 | −0.45 | 9.21 × 10−2 |
Complex IV | |||
LOC100055813 | cytochrome c oxidase subunit 6C | 1.86 | 5.20 × 10−2 |
COX6A2 | cytochrome c oxidase subunit 6A2 | 1.81 | 4.75 × 10−2 |
CYC1 * | cytochrome c1 heme protein | 0.67 | 4.75 × 10−2 |
Complex V | |||
ATP5MG | ATP synthase subunit g | 1.24 | 4.56 × 10−2 |
ATP5PF * | ATP synthase-coupling factor 6 | 1.07 | 4.75 × 10−2 |
ATP5F1D | ATP synthase subunit delta | 0.73 | 9.00 × 10−2 |
ATP5F1B | ATP synthase subunit beta | 0.43 | 9.39 × 10−2 |
SLC25A4 * | ADP/ATP translocase 1 | 0.52 | 8.84 × 10−2 |
Fat metabolism | |||
HADHB | trifunctional enzyme subunit beta | 1.22 | 4.75 × 10−2 |
TCA cycle | |||
CS * | citrate synthase | 0.61 | 5.20 × 10−2 |
SUCLA2 * | Succinate—CoA ligase subunit beta | 0.88 | 5.20 × 10−2 |
IDH3B * | isocitrate dehydrogenase [NAD] subunit beta isoform X2 | 0.77 | 4.75 × 10−2 |
FH * | fumarate hydratase | −0.51 | 6.19 × 10−2 |
Other mitochondrial | |||
SOD2 | superoxide dismutase [Mn] mitochondrial precursor | 1.15 | 4.75 × 10−2 |
VDAC2 | voltage-dependent anion-selective channel protein 2 | 0.85 | 5.20 × 10−2 |
PPA2 * | Cluster of inorganic pyrophosphatase 2 | 0.53 | 9.21 × 10−2 |
MRPS36 * | 28S ribosomal protein S36 | 0.54 | 8.43 × 10−2 |
Protein Name | Log2FC | Adj. P | |
---|---|---|---|
Sarcomere | |||
Thick and thin filaments | |||
MYL1 | myosin light chain 1/3 skeletal muscle isoform isoform X1-fast-twitch | 1.91 | 4.75 × 10−2 |
TNNI1 | troponin I slow-twitch | 1.43 | 4.75 × 10−2 |
TPM3 | tropomyosin alpha-3 chain isoform X4 slow-twitch | 0.93 | 4.75 × 10−2 |
LOC100062893 | myosin 8 | 0.79 | 5.20 × 10−2 |
TPM1 | tropomyosin alpha-1 chain isoform X5 | 0.71 | 6.55 × 10−2 |
MYOM1 | Cluster of myomesin-1 | 0.69 | 5.20 × 10−2 |
MYL3 | myosin light chain 3-slow twitch | 0.67 | 6.19 × 10−2 |
TNNI2 | troponin I fast twitch | 0.62 | 8.77 × 10−2 |
MYBPH | myosin-binding protein H | −0.53 | 7.31 × 10−2 |
Z disc | |||
CSRP3 | cysteine and glycine-rich protein 3 | 1.16 | 4.55 × 10−2 |
FHL1 | four and a half LIM domains protein 1 isoform X3 | 1.13 | 4.75 × 10−2 |
MYOT | myotilin isoform X1 | 1.01 | 8.74 × 10−2 |
PDLIM3 | Cluster of PDZ and LIM domain protein 3 | 1.00 | 4.56 × 10−2 |
PDLIM5 | PDZ and LIM domain protein 5 isoform X9 | 0.87 | 5.20 × 10−2 |
DES | desmin | 0.79 | 4.75 × 10−2 |
MYOZ1 | myozenin-1 fast-twitch | 0.68 | 4.75 × 10−2 |
SYNPO2 | synaptopodin-2 | 0.63 | 6.19 × 10−2 |
Metabolism | |||
Polysaccharides | |||
AKR1B1 | aldose reductase | 1.19 | 6.19 × 10−2 |
ENO2 | gamma-enolase | 1.18 | 5.26 × 10−2 |
PGAM2 | phosphoglycerate mutase 2 | 0.75 | 8.74 × 10−2 |
ALDOC | fructose-bisphosphate aldolase C | 0.63 | 6.19 × 10−2 |
PYGM | myophosphorylase | 0.47 | 8.47 × 10−2 |
GYG | glycogenin 1 | 0.55 | 8.74 × 10−2 |
Protein | |||
GOT1 * | aspartate aminotransferase cytoplasmic | 0.78 | 6.19 × 10−2 |
PADI2 | protein-arginine deiminase type-2 | −0.60 | 6.55 × 10−2 |
Lipid | |||
PGP | glycerol-3-phosphate phosphatase | −0.48 | 9.18 × 10−2 |
Antioxidant | |||
PRDX2 * | Cluster of peroxiredoxin-2 | −0.58 | 5.20 × 10−2 |
CAT | Cluster of catalase | −0.59 | 8.19 × 10−2 |
Heat Shock factors | |||
HSPA8 | heat shock cognate 71 kDa protein | 0.79 | 4.75 × 10−2 |
HSPA1A | heat shock 70kDa protein 1A | 0.48 | 9.39 × 10−2 |
Inflammation | |||
RTN4 | reticulon-4 isoform X4 | 1.59 | 4.56 × 10−2 |
MIF | macrophage migration inhibitory factor | −0.70 | 4.75 × 10−2 |
Other | |||
MYH15 | myosin-15 | 1.75 | 2.41 × 10−2 |
AHSG | alpha-2-HS-glycoprotein | 1.15 | 9.88 × 10−2 |
SPI2 | alpha-1-antiproteinase 2 isoform X1 | 1.15 | 7.51 × 10−2 |
LOC100065068 | alpha-1-antiproteinase 2-like precursor | 0.88 | 8.47 × 10−2 |
EEF1A2 | elongation factor 1-alpha 2 | 0.73 | 6.19 × 10−2 |
PEBP1 | phosphatidylethanolamine-binding protein 1 | 0.65 | 7.50 × 10−2 |
YWHAE | Cluster of 14-3-3 protein epsilon | 0.58 | 6.19 × 10−2 |
EHD2 | EH domain-containing protein 2 | −0.52 | 7.97 × 10−2 |
APOA1 | apolipoprotein A-I | −0.58 | 4.75 × 10−2 |
HBA | hemoglobin subunit alpha | −0.61 | 9.11 × 10−2 |
F2 | prothrombin | −0.65 | 4.66 × 10−2 |
CCT6A | T-complex protein 1 subunit zeta | −0.69 | 4.75 × 10−2 |
ILK | integrin-linked protein kinase isoform X2 | −0.70 | 6.19 × 10−2 |
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Valberg, S.J.; Velez-Irizarry, D.; Williams, Z.J.; Henry, M.L.; Iglewski, H.; Herrick, K.; Fenger, C. Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis. Genes 2022, 13, 1853. https://doi.org/10.3390/genes13101853
Valberg SJ, Velez-Irizarry D, Williams ZJ, Henry ML, Iglewski H, Herrick K, Fenger C. Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis. Genes. 2022; 13(10):1853. https://doi.org/10.3390/genes13101853
Chicago/Turabian StyleValberg, Stephanie J., Deborah Velez-Irizarry, Zoë J. Williams, Marisa L. Henry, Hailey Iglewski, Keely Herrick, and Clara Fenger. 2022. "Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis" Genes 13, no. 10: 1853. https://doi.org/10.3390/genes13101853
APA StyleValberg, S. J., Velez-Irizarry, D., Williams, Z. J., Henry, M. L., Iglewski, H., Herrick, K., & Fenger, C. (2022). Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis. Genes, 13(10), 1853. https://doi.org/10.3390/genes13101853