Reasons for performing study: MicroRNAs (miRNA) are small endogenous noncoding interfering RNA molecules (18–25 nucleotides) regarded as major regulators in eukaryotic gene expression. They play a role in developmental timing, cellular differentiation, signalling and apoptosis pathways. Because of the central function of miRNAs in the proliferation and differentiation of the myoblasts demonstrated in mouse and man, it is assumed that they could be present in equine muscles and their expression profile may be related to the muscle status.
Objective: To identify miRNA candidates in the muscles of control and affected horses suffering from polysaccharide storage myopathy (PSSM) and recurrent exertional rhabdomyolysis (RER).
Methods: Muscle biopsies were collected in the gluteus medius of horses allocated into 4 groups: French Trotters (3 control-TF vs. 3 RER-TF) and Norman Cob (5 control-Cob vs. 9 PSSM-Cob). Blood samples were collected for miRNA analysis. Total RNA were extracted and real time quantitative RT-QPCR analysis were conducted using 10 miRNA assays (mir-1-23-30-133-181-188-195-206-339-375).
Results: All the miRNA candidates were significantly detected in the muscles and some in blood samples. Variance analysis revealed highly significant (P<0.0001) effects of the miRNA type, breed and pathology on the miRNA expression. A specific miRNA profile was related to each myopathy: a higher expression of mir-1, 133, 23a, 30b, 195 and 339 in RER-TF vs. control-TF (P<0.05); a higher expression of mir-195 in PSSM-Cob vs. control-Cob (P<0.05). The miRNA profile was different between breeds for mir-181, 188 and 206 (P<0.05). The mir-1, 133, 181, 195 and 206 were detected in blood of control-Cob and PSSM-Cob horses.
Conclusions: This first study about muscular miRNA profile in equine myopathies indicated that it is possible to discriminate pathological from control horses according to their miRNA profile. The RER miRNA profile was more specific and contrasted than the PSSM profile.
MicroRNA (miRNA) are small endogenous noncoding interfering RNA molecules (18–25 nucleotides) regarded as major post transcriptomic regulators in eukaryotic gene expression. They were discovered in C. elegans (small RNA lin-4) (Lee et al. 1993) and then in Arabidopsis plants (Llave et al. 2002). Multiple eukaryotes including fungi, plants, protozoans and metazoans produce RNA silencing systems involved in many gene regulation processes (Carrington and Ambros 2003). Bio-informatic studies have predicted that about 30% of the genes have a target sequence where a miRNA could hybridise to inhibit the translation of the mRNA (Filipowicz et al. 2008). mRNA, highly conserved between distantly related species, play an important role in developmental timing, cellular differentiation, signalling and apoptosis pathways.
In human skeletal muscle, about 10 miRNA were identified to have important functions in muscle development, maintenance and healing. The miRNA were involved in the control of myoblast proliferation and differentiation in myotubes. The essential myo-miRNA are the following: mir-1, mir-23a, mir-30b, mir-133, mir-181, mir-188, mir-195, mir-206, mir-339 and mir-375 (Anderson et al. 2006; Chen et al. 2006; Clop et al. 2006; McCarthy et al. 2007a,b). In addition, the mir-1, mir-133 and mir-181 have several isoforms with a few nucleotide differences located out of the seed sequence. Other miRNA could be suspected to have a function in the muscle differentiation: mir-100, mir-191, mir-138-2, mir-22 because they are predicted targets of MyoD and myogenin (Rao et al. 2006).
Because of the central function of miRNA in muscle development, we assumed that they could be present in equine muscles and may be in the blood. Their expression profile should be related to muscle physiology or pathology. For this first miRNA study, we investigated the 2 more frequent equine myopathies in Warmblood horses (Hunt et al. 2008): polysaccharide storage myopathy (PSSM) and recurrent exertional rhabdomyolysis (RER). The purpose of this study was to identify miRNA candidates in the muscles of control and affected horses suffering from PSSM and RER in 2 different breeds. A complementary objective was to test the possibility to detect some of the muscular miRNA in the blood.
Materials and methods
Horses and muscle sample collection
A total of 20 horses were allocated into 4 groups according to their breed and muscle pathology:
• Norman Cob (Cob): control-Cob (5) vs. PSSM-Cob (9)
• French Trotters (TF): control-TF (3) vs. RER-TF (3)
All the PSSM horses were genotyped for the GYS1 c.926G>A mutation (McCue et al. 2008). Most of the horses were heterozygous (GA) and one was homozygous (AA). All the Cob horses were under the same conditions of nutrition and low draught exercise activity on a studfarm. However, the PSSM horses had a low fitness level. The PSSM horses suffered from chronic rhabdomyolysis as reported in Barrey et al. (2009).
In the TF trotters suffering from RER, clinical signs of muscle stiffness (‘tying-up’) and pain were observed by the trainers during a training session. Muscle biopsies and blood samples were collected as soon as possible within 24 h after the ‘tying-up’ signs. The blood samples were collected for biochemistry analysis of CKM (6900–24 500 UI/l) and ASAT (730–15 250 UI/l). All the RER Trotters were under middle intensity training and belonged to different stables in the same training centre (Grosbois, Boissy Saint Léger, France). The control Trotters were fit but not in training at the time of muscle biopsy.
For each horse, a microbiopsy of the gluteus medius muscle was performed under local anaesthesia. This muscle was chosen because of: (i) its important propulsion function in horse locomotion, (ii) its early and major histological involvement in the course of PSSM (Larcher et al. 2008) or RER disease (Valberg et al. 1993) and (iii) the good standardisation of biopsy sampling (Valette et al. 1999). The horses were sedated and received a local anaesthesic before a small skin incision was made according to a standardised protocol used routinely for muscle disease diagnostis. The biopsy of the dorsal compartment of the gluteus medius was performed at the first third distance between the sacral and coxae tuber. The biopsy needle with automatic sampling was vertically inserted until its extremity was just under the fascia corresponding to a 3.5 cm depth for the gluteus medius. Blood was removed from the muscle sample by absorbing it with a compress. The muscle sample was put in the RNA stabiliser for 1 h (RNA later1) and then frozen at −80°C until analysis. In order to investigate the possibility of detecting miRNA in the serum or plasma, a preliminary trial using the different types of blood collection tubes (dry, EDTA, heparin, fluorure and RNA stabiliser) (PAXgene blood RNA Kit)2 was undertaken. Finally, blood samples were collected in 7 Cob horses still available at the moment of the miRNA study (5 PSSM and 2 controls) on PAXgene blood RNA tubes.
Informed consent was obtained from the horse owner and the study was approved by the animal care committee of the National Research Institute of Agriculture (INRA).
Total RNA was extracted from the muscle samples by a phenol-chloroform method developed by Chomczynski and Sacchi (1987) (Trizol reagent)3. Briefly, the reagent, a monophasic solution of phenol and guanidine isothiocyanate, maintained the integrity of the RNA while disrupting the cells and dissolving components. The addition of chloroform followed by centrifugation, separated the solution into an aquaeous and organic phase. RNA remained exclusively in the supernatant aqueous phase and the DNA and proteins remained in the heavy organic phase.
The total RNA of the blood samples was extracted according to the manufacturer's protocol (PAXgene® blood RNA Kit)2. In order to avoid DNA contamination, a DNase digestion and a total RNA purification by filtering were applied after an initial proteinase digestion. The other blood sample tubes were centrifuged to collect the serum for total RNA extraction by the phenol-chloroform method (Trizol reagent)3.
The total RNA concentration and RNA purity were measured by optical density with a spectrophotometer at 260 nm and the ratio 260/280 (Nanodrop)4. RNA was considered as pure when the 260 nm over 280 nm absorbance ratio was close to 2. The quality of total RNA was verified by micro channel electrophoresis (RNA 6000 Nano LabChip, Bioanalyzer)5. The RNA were stored at −80°C.
The expression levels of 10 miRNA were measured using human microRNA assays including a reverse transcription stem-loop followed by a real time PCR for quantitation of specific mature miRNA (TaqMan microRNA assays)6. This method has been validated as a very sensitive (25 pg total RNA) and very specific method (Chen et al. 2005). It does not accept any mismatch in the short sequence of the miRNA. The 10 microRNA assays used were selected because of their muscular activity already described in the literature: mir-1, mir-23a, mir-30b, mir-133, mir-181, mir-188, mir-195, mir-206, mir-339 and mir-375 (Anderson et al. 2006; Chen et al. 2006; Clop et al. 2006; McCarthy et al. 2007a,b). In addition, an alignment of the human mature miRNA sequence was performed using a BLASTN method on the equine genome to verify the perfect match of the 2 sequences. Quantitation of each miRNA and a reference microRNA panel (all the human miRNA sequences described in miRBase 9; Griffiths-Jones et al. 2006) taken as a positive control, was performed in 2 steps according to the manufacturer's instructions: 1) in the reverse transcription step, cDNA was reverse transcribed from 10 ng total RNA of each sample using specific stem-loop primer and reagents from the reverse transcription kit (TaqMan MicroRNA Reverse Transcription Kit)6; the quantity of each RNA sample input was measured by optical density with a spectrophotometer (Nanodrop)4 and 2) in the real-time PCR step, RT products were amplified from cDNA samples diluted to one third together with the PCR mix (Universal PCR master Mix)6. The microRNA specific probes contained a reporter dye (FAM), a minor groove binder and a no fluorescent quencher. Each miRNA of each sample was replicated. The Ct results were averaged after removing the potential outliers. In order to calculate relative miRNA expression in the control groups and the pathological groups, we used the delta Ct method: miRNA relative expression (fold changes) =ΔΔCt. The housekeeping miRNA was chosen for its stability under the different conditions (breeds and pathology). According to the following results, mir-375 was the more adapted housekeeping miRNA. For the pathological groups, each miRNA expression was relative to the reference miRNA that was the corresponding miRNA of the control group. For the blood miRNA real time RT-PCR analysis, the relative expression of the miRNA of PSSM horses was calculated using mir-195 as housekeeping miRNA and the corresponding miRNA of the control-Cob group as reference miRNA.
Descriptive statistics were used to present the raw results of real time quantitative RT-PCR. In order to compare the miRNA type (10 miRNA), breed (COB vs. TF) and muscle pathology (RER, PSSM vs. control) effect on the expression values in the muscle sample, a 3 ways variance analysis was applied to test the effect of miRNA type (10 levels), breed (TF or COB) and pathology (control, PSSM, RER) (GLM, NCSS 2007)7. If a significant effect at P<0.05 was detected, a mean comparison test was applied to compare the means of the subgroups i.e. the 10 miRNA levels, TF vs. Cob, RER vs. control-TF and PSSM vs. control-Cob.
Finally, in order to determine if the miRNA profile of each horse could be used to classify them according to their breed and pathology (4 groups), all the data were analysed using a discriminant analysis (NCSS 2007)7. Briefly, a discriminant analysis is a statistical method for classifying a set of observations into predefined groups (here the 4 groups 2 breeds times 2 status). In the present study, each sample was described by 10 miRNA values, taken as variables. The goal of the method is to maximise the variance between the classes by computing a set of new canonical variables (scores 1, 2, 3) as linear combination of the measured variables (the 10 miRNA levels).
After averaging the replicates, a total of 173 real time quantitative RT-PCR results were obtained on the muscle samples in addition to the positive and negative controls. All miRNA were detected in the muscles with a mean cycle threshold comprised between 19.37 (mir and 133) up to 38.8 (mir-375). Figure 1 shows the amplification plot obtained during the real time quantitative RT-PCR for one set of analysis. Negative controls (water) and positive controls (panel of synthetic miRNA) were used to assess the quality of the real time quantitative RT-PCR results expressed by the PCR cycle threshold (Ct). The negative control had undetermined Ct because the cycle threshold was over 40 (maximum number of PCR cycles). The positive controls reached high Ct values (mean Ct [s.d.]= 16.06 [0.86]) because of the high concentration of most of the synthetic miRNA included in this panel. The low variability (Coefficient of variation % = 0.86/16.06 = 5%) of the Ct positive control indicated the good standardisation of different assays. One unit of Ct corresponds to double the miRNA concentration. Figure 2 illustrates the general miRNA profile in the control horses of the 2 breeds. There was a great variability of expression between the miRNA profiles in the 2 breeds. The Ct values of the more expressed miRNA mir-1, mir-133 and mir-30b are between 18 and 22. The mir-375 had a low variability and expression level in both groups (2 breeds and pathological groups). Consequently, this mir-375 was chosen as the housekeeping miRNA in the following relative expression calculations presented in Table 1.
Table 1. Results of the 3 way analysis of variance to test simultaneously the miRNA type, pathology type and breed effect. The relative expressions of the miRNA expressed in fold changes were calculated for the pathological groups using the corresponding miRNA of the control group as reference miRNA and mir-375 as housekeeping miRNA. On the right side column (miRNA means comparison), the Fisher least square difference multiple-comparison test compared the means of the miRNA expressions in the pathological group vs. control group within each breed. The very low level of miRNA expression that was considered as undetermined was set to Ct=40 in order to calculate relative expression and discriminant analysis
miRNA means comparison
Relative miRNA expression (fold changes)
Breed effect P<0.00001
miRNA effect P<0.000001
Pathology effect P<0.0001
The 3 way variance analysis revealed high significant effects of the miRNA type (F ratio = 69.26; P<0.000001), breed (F ratio = 21.23; P<0.00001) and pathology (F ratio = 7.38; P<0.001) on the miRNA expression values (Table 1). The miRNA profiles observed in each myopathy were different. The averaged miRNA expressions of RER horses (Ct = 21.38 ± 0.37) were significantly higher than in PSSM horses (Ct = 30.23 ± 0.19). When the pathology effect was significant at P<0.05, a mean comparison test was applied to compare the means between control and pathological groups (Table 1 last column on the right side). In RER horses, many miRNA exhibited a great upregulation between 44- to 5673-fold changes: mir-1, mir-133, mir-23a, mir-30b, mir-195 and mir-339 (Table 1). In PSSM horses, only 2 moderate miRNA expression modifications were observed: a 0.14-fold down regulation of mir-1 and a 6.8-fold upregulation of mir-195 (P<0.05) (Table 1). In the homozygous PSSM horse (AA), some miRNA were more upregulated than in the other PSSM horses. The following miRNA relative expressions against the control-Cob horses were calculated: mir-195 (114-fold), mir-206 (29-fold), mir-188 and mir-23a (6-fold) and mir-181 (5-fold). The averaged miRNA expressions were higher in the Cob (Ct = 24.36 ± 0.15) than in the French Trotters (Ct = 30.10 ± 0.25). The miRNA expressions of mir-181, mir-188 and mir-206 were higher in the control-Cob than the control-French Trotter (P<0.05).
The discriminant analysis correctly classified 93.3% of the horses according to their group (breed pathology) using their miRNA profile (10 miRNA expression values). Only one misclassification was observed for a control Cob horse which has been classified as a PSSM case. The 2 first canonical variables (Scores 1 and 2) could explain almost all the total variance of the data (99.8%). Figure 3 shows the results of this multivariate analysis in a 3 dimensional space. The first plane (Scores 1 and 2) better discriminated the RER from the control-TF horses (maximal variance: 70.3% of total variance). In the second and third planes (Scores 1 and 3 and Scores 2 and 3), the analysis better discriminated the PSSM horses from the control-Cob horses (lower part of the total variance: 29.4%).
The miRNA can be detected in equine serum and plasma (Table 2). According to our preliminary trials on blood collection tubes and miRNA analysis, the expression level in the serum was lower than in the plasma or total blood. The best collecting tube for miRNA analysis was the tube including 5 ml of RNA stabiliser (Paxgene blood RNA kit)2 or dry tube. The miRNA expressions (mir-133, mir-181, mir-195, mir-206) were comprised between 34.95 and 38.51 in PSSM horses while there was a tendency of lower expression in the control-Cob horses (Table 2). The expressions of mir-1 and mir-206 were very low in both groups. In the homozygous PSSM horse (AA), it was observed that the following miRNA were more upregulated than in the control-Cob horses: mir-206 (10-fold), mir-181 (3.2-fold) and mir-195 (2.8-fold).
Table 2. MiRNA expression levels in the blood of control and PSSM Cob horses expressed as cycle threshold (Ct). The relative expressions of the miRNA expressed in fold changes were calculated for the pathological groups using the corresponding miRNA of the control group as reference miRNA and mir-195 as housekeeping miRNA. There was no significant difference of miRNA expressions between the 2 groups. However, a tendency of a small upregulation in the PSSM horses was observed in one homozygous PSSM horse
Blood of control-Cob (Ct)
Blood of PSSM Cob (Ct)
Relative expression (fold changes)
Significant concentrations of 10 microRNA were found in the control and pathological equine muscle. These small RNA were 18–25 nucleotides long and identified in the total muscular RNA using real-time quantification by stem-loop RT-PCR. This method has been validated as a very sensitive (25 pg total RNA) and very specific method (Chen et al. 2005). It does not accept any mismatch in the short sequence of the mature miRNA. The relative miRNA expression calculation using the delta Ct method with a reference and housekeeping miRNA was applied in the present study. However, the housekeeping miRNA candidates are not yet well described in the literature. According to the results, we decided to use mir-375 as housekeeping miRNA for muscle and mir-195 for blood results. They were the 2 more stable miRNA, independent of breed or pathology effect.
It should be underlined that all the miRNA found in the equine muscle were already known as human miRNA (miRBase, Griffiths-Jones et al. 2006). We confirmed the similarity of the human and equine mature miRNA sequences using a BLASTN method on the equine genome to verify the perfect match of the 2 sequences. In addition, these myo-miRNA are well conserved among species and were predicted in horses by a bio-informatic study (Zhou et al. 2009). These results confirmed the high conservative sequence of the miRNA among mammalian species as shown for other important miRNA: mir-433 and mir-127 (Song and Wang 2009). The present study gives so far the first experimental results demonstrating the presence of miRNA in equine muscle and also finding significant miRNA expression levels in the blood and serum. One other study in human patients suffering from cancer has demonstrated the presence of significant miRNA concentrations in the serum and plasma (Chen et al. 2008). These authors verified that miRNA were also detected in animal serum such as mice, rats, cattle and horses where they identified let-7a, mir-21, mir-223, mir-451, mir-24 and mir-20a by semi-quantitative RT-PCR. The blood miRNA comes from other tissues after cell lysis and from leucocytes. The microRNA are very stable in the blood stream because of their hairpin structure that protects them against RNAse activity. Finally, the total number of miRNA in man is about 721 which gives many profile combinations related to a physiological or pathological status. In horses, 346 miRNA were predicted in silico (Zhou et al. 2009) but many others will be discovered. In the near future, miRNA profile in blood and other tissues will become new type of disease biomarkers.
The horse genome has been recently sequenced and published (Horse genome project 2007; http://www.ncbi.nlm.nih.gov/genome/guide/horse/; Wade et al. 2009) allowing the use of the horse genome sequence to predict the microRNA candidates in in silico studies (Zhou et al. 2009). They blasted all the animal miRNA known in the data bases (miRBase) and predicted 354 novel mature miRNA specific of the horse. These miRNA should be organised in clusters and were more concentrated in the chromosomes 11, 24 and X. In the present study we experimentally identified 10 miRNA involved in muscular development and healing in mice, rats or man. It was observed that the expression levels of most of the miRNA were higher in pathological horses. Much higher expression values (mir-133 > mir-1 > mir-23a > mir-195) were observed in RER than PSSM horses (mir-195). In addition, a first observation showed that the miRNA expression could be detected in blood both in PSSM and control horses. The homozygous PSSM horse (AA for GYS mutation) included in the PSSM group expressed a higher upregulation of mir-195, mir-206, mir-188, mir-23a, and mir-181 in the muscle and mir 206, 181 and 195 in the blood. However, further investigations should be conducted to confirm the tendency observed in this preliminary observation in a larger group of homozygous PSSM horses.
In the RER group, the miRNA upregulation may be related to the proliferation and differentiation that took place in the muscular healing process. The function of each miRNA can be briefly discussed. Figure 4 illustrates the state of the knowledge of the proliferation and differentiation regulation of the myoblasts by the main myo-miRNA in humans: mir-133, mir-1, mir-181 and mir-206. MyoD, myogenin, SRF and MEF2C are the 4 essential myogenic transcription factors. These transcription factors stimulate the transcription of 4 miRNA and specific genes of muscle proteins. In addition, it seems that there is a feedback control of their own transcriptions by mir-133 (Rao et al. 2006). For the first step of proliferation of the myoblast, miRNA-133 is upregulated and inhibits the serum response factor (SRF). SRF is known to be an important transcription factor in adults and a decrease of this factor is related to a premature ageing of muscle loss (Lahoute et al. 2008). Mir-375 could regulate myotrophine transcription. The high level of expression of mir-133 in RER muscle could be related to the stimulation of the myoblast proliferation after rhabdomyolysis of some muscle fibres. In Cob horses, mir-133 was highly expressed in both control and pathological groups and was detected at a low level in the blood.
For the second step of differentiation in myotubes, 3 miRNA are up regulated: mir-1, mir-181 and mir-206. Mir-181 is required but not sufficient for differentiation (Naguibneva et al. 2006). The concentration of this miRNA is very low at the end of myotube differentiation but increases mainly for muscle fibre regeneration after rhabdomyolysis or injury. Mir-206 is more involved in the maturation process of the myotubes (Yuasa et al. 2008a). It inhibits utrophin which is exchanged by dystrophine and maybe it inhibits myosin heavy chain II (MHC II) and consequently releases the expression of MHC I (McCarthy et al. 2007b; Yuasa et al. 2008b).
Again mir-1 and mir-181 were more expressed in the RER muscle than the control-TF horses. In the Cob horses, the 3 mir-1, mir-181 and mir-206 were highly expressed in both groups.
In summary, the RER muscles show a miRNA profile which could be related to the proliferation and differentiation regulation of the myoblasts activated after rhabdomyolysis. In addition, the contrast of the expression level in pathological groups vs. controls is higher in RER than in PSSM horses. The miRNA upregulations were more pronounced in the acute rhabdomyolysis observed in RER than in the chronic rhabdomyolysis in PSSM.
The results detected a significant difference in the miRNA expression of the 2 breeds. The averaged miRNA expression was higher in the Cob control group than in the French Trotter control group. This result could be related to the breed genetic effect and also to the exercise and training effect. The French Trotters were more trained and performed regular intense exercises. The Cob horses were less trained and performed low intensity draft exercises.
The use of miRNA profile in tissues and blood for the disease diagnostic is under a quick development in human cancerology and it has been evaluated in myology (Calin and Croce 2006; Chen et al. 2008). In the major human myopathies, many miRNA (151) were upregulated and only 28 were downregulated among 428 miRNA measured in the pathological muscles (Eisenberg et al. 2007). The general trends of the present study were in agreement with this human study especially for the RER muscle where several miRNA, like mir-133, mir-1 and mir-23a were highly upregulated while in PSSM muscle only mir-195 was moderately upregulated.
As in other eukaryotes, many microRNA can be quantified in the equine muscle and can be detected in the blood stream where they are stable. This first study about muscular miRNA profile in equine myopathies indicates that it is possible to discriminate pathological from control horses according to their miRNA profile using only 10 miRNA candidates. The RER miRNA profile was more contrasted and specific with several highly up-regulated miRNA (mir-133, mir-1, mir-23a) than the PSSM miRNA profile (mir-195). Further investigations will be required to chararacterise the specificity and sensitivity of theses new genomic biomarkers of myopathies both in muscle and blood samples.
Conflicts of interest
The authors declare no potential conflicts.
This study was supported jointly by the Institut de la Recherche Agronomique (INRA), Institut National de la Santé et de la Recherche Médicale (INSERM) and Genopole Evry. Mrs Wendy Brand-Williams (INRA, genetic Department) is greatly acknowledged for her English revision.
1RNA later, Ambion, Ambion operates as a subsidiary of Life Technologies Corporation. Les Ulis, 91943 Courtaboeuf, France.
2 BD and Qiagen, Germany.
3 GibcoBRL, Life technologies, USA.
4 Thermo scientific, Delaware, USA.
5 Bioanalyzer, Agilent Technologies, 91745 Massy, France.
6 Applied Biosystems, Applied Biosystems operates as a subsidiary of Life Technologies Corporation. Les Ulis, 91943 Courtaboeuf, France.