Transcriptome analysis of muscle in horses suffering from recurrent exertional rhabdomyolysis revealed energetic pathway alterations and disruption in the cytosolic calcium regulation.

Recurrent exertional rhabdomyolysis (RER) is frequently observed in race horses like trotters. Some predisposing genetic factors have been described in epidemiological studies. However, the exact aetiology is still unknown. A calcium homeostasis disruption was suspected in previous experimental studies, and we suggested that a transcriptome analysis of RER muscles would be a possible way to investigate the pathway disorder. The purpose of this study was to compare the gene expression profile of RER vs. control muscles in the French Trotter to determine any metabolic or structural disruption. Total RNA was extracted from the gluteal medius and longissimus lumborum muscles after biopsies in 15 French Trotter horses, including 10 controls and 5 RER horses affected by 'tying-up' with high plasmatic muscular enzyme activities. Gene expression analysis was performed on the muscle biopsies using a 25K oligonucleotide microarray, which consisted of 24,009 mouse and 384 horse probes. Transcriptome analysis revealed 191 genes significantly modulated in RER vs. control muscles (P < 0.05). Many genes involved in fatty acid oxidation (CD36/FAT, SLC25A17), the Krebs cycle (SLC25A11, SLC25A12, MDH2) and the mitochondrial respiratory chain were severely down-regulated (tRNA, MT-ND5, MT-ND6, MT-COX1). According to the down-regulation of RYR1, SLC8A1 and UCP2 and up-regulation of APP and HSPA5, the muscle fibre calcium homeostasis seemed to be greatly affected by an increased cytosolic calcium and a depletion of the sarcoplasmic reticulum calcium. Gene expression analysis suggested an alteration of ATP synthesis, with severe mitochondrial dysfunction that could explain the disruption of cytosolic calcium homeostasis and inhibition of muscular relaxation.

Gene expression profiling in equine muscle tissues using mouse cDNA microarrays.

REASONS FOR PERFORMING STUDY: Progress could be achieved by using microarrays to understand metabolic adaptations and disorders in equine muscle in response to exercise. OBJECTIVES: To test the feasibility of using mouse cDNA microarrays to analyse gene expression profile in normal equine muscles. METHODS: Muscular biopsies of dorsal gluteus medius and longissimus lumborum were done in 4 healthy Standardbreds. Total RNA was extracted from the muscle samples. The concentration and quality of RNA were measured before and after amplification. Gene expression profiles were measured using mouse cDNA microarrays including 15,264 unique genes representing about 11,000 documented genes. Three hybridisation tests were performed to check interspecificity, reproducibility and to compare gene expression in these muscles. For each test, a dye-swap hybridisation with Cy3 and Cy5 fluoromarkers were done and the gene list filtered according the signal level. RESULTS: According to the specificity test, the mouse cDNA microarrays were correctly hybridised by equine muscle cDNA. All positive control genes (GAPDH, HPRT and beta-Actin) and no negative control gene (yeast, plant) hybridised. The reproducibility test demonstrated a good linearity between the duplicate hybridisations: 99.99% of the significant expressed genes have an expression ratio between 1.4 and 1/1.4 = 0.71. These limits can be considered as the thresholds to qualify as up-regulated (ratio >1.4) or downregulated (ratio <0.71). In the muscle comparison test between gluteus medius vs. longissimus lumborum, 63 genes were found up-regulated and 8 genes down-regulated. The range of gene expression ratios in the gluteus medius was 0.61-8.31 x the longissimus lumborum. This list of modulated genes was classified by functions using a gene ontology data basis. CONCLUSION: Mouse microarrays could be used to hybridise equine RNA extracted from muscle tissues. For many genes there are large sequence identities that allowed interspecific cDNA hybridisation. The sensitivity of the method allowed quantification of up- and down-regulated genes after applying appropriate filters. POTENTIAL RELEVANCE: Expression profiling could be used to explore the muscle metabolism changes related to exercise, training, pathology and illegal medication in horses.