Reasons for performing study: In exercising horses, up to 50% of blood lactate is taken up into red blood cells (RBCs). Lactate transporter proteins MCT1, MCT2 and CD147 (an ancillary protein for MCT1) are expressed in the equine RBC membrane. In Standardbreds (SB), lactate transport activity is bimodally distributed and correlates with the amount of MCT1 and CD147. About 75% of SB studied have high lactate transport activity in RBCs. In other breeds, the distribution of lactate transport activity is unknown.
Objectives: To study whether similar bimodal distribution of MCT1 and CD147 is present also in the racing Finnhorse (FH) and Thoroughbred (TB) as in the SB and to study the distribution of MCT2 in all 3 breeds and to determine if there is a connection between MCT expression and performance markers in TB racehorses.
Methods: Venous blood samples were taken from 118 FHs, 98 TBs and 44 SBs. Red blood cell membranes were purified and MCT1, MCT2 and CD147 measured by western blot. The amount of transporters was compared with TB performance markers.
Results: In TBs, the distribution of MCT1 was bimodal and in all breeds distribution of MCT2 unimodal. The amount of CD147 was clearly bimodal in FH and SB, with 85 and 82% expressing high amounts of CD147. In TBs, 88% had high expression of CD147 and 11% low expression, but one horse showed intermediate expression not apparent in FH or SB. Performance markers did not correlate with the amount of MCT1, MCT2 or CD147.
Conclusions: High lactate transport activity was present in all 3 racing breeds, with the greatest proportion in the TB, followed by the racing FH, then SB. There was no significant statistical correlation found between lactate transporters in RBC membrane and markers of racing performance in the TB.
During intense exercise, muscles depend on anaerobic metabolism to produce ATP, which results in lactate accumulation in muscles. Lactate-induced acidification is one of the major factors contributing to muscle fatigue (Juel 2008). At physiological pH, lactic acid is mostly in a dissociated form and needs a transporter to cross the muscle cell membrane into plasma. During exercise most lactate is transported by monocarboxylate transporters (MCT), which transport monocarboxylate anions, such as lactate, together with protons across cell membranes (Juel 2008). The transport is controlled by hydrogen ion gradients and does not require ATP (Merezhinskaya and Fishbein 2009). The MCT family includes at least 14 members, of which isoforms 1–4 are known to transport lactate (Halestrap and Meredith 2004). Of these proteins, MCT1 and MCT4 need an ancillary protein, CD147 (EMMPRIN, basigin, neurothelin), for translocation to the plasma membrane and also to form an active transporter complex (Kirk et al. 2000; Gallagher et al. 2007).
Previously, lactate transport activity has been studied only in the Standardbred. In this breed, about 75% of the population studied has high MCT1 lactate transport activity (HT) in the RBC and the rest of the population has low lactate transport activity (LT; Väihkönen and Pösö 1998). In vivo, it has been shown that RBC lactate concentration is higher in horses with high lactate transport activity than in horses with low lactate transport activity after submaximal and maximal exercise (Väihkönen et al. 1999; Koho et al. 2002). An indirect connection between lactate transportactivity and performance was demonstrated by Räsänen et al. (1995), who showed that horses with higher lactate concentration in their RBCs were better performers. In the Standardbred, low lactate transport activity has been shown to be inherited as an autosomal recessive trait (Väihkönen et al. 2002).
To study whether similar bimodal distribution of MCT1 and CD147 is present also in breeds other than the Standardbred, we measured the amount of CD147 and MCT1 in RBC membranes of the racing Finnhorse and Thoroughbred. Finnhorses represent cold-blooded trotters, that race in similar conditions, as Standardbreds. In contrast, Thoroughbreds are hot-blooded and have been bred for centuries for more sprint type performance. We also measured the amount of MCT2 in all 3 breeds. Furthermore, we compared performance parameters with the amount of lactate transporting proteins in Thoroughbred racehorses to see if high lactate transporting capability might be beneficial in exercise.
Materials and methods
This study used 118 Finnhorses, 98 Thoroughbreds and 44 Standardbreds (Table 1). The horses were clinically healthy. EDTA-blood samples were collected with owner's consent from the jugular vein, centrifuged and red blood cells separated. Red blood cells were stored at -80°C until analysed. The study complied with the ethics policy for animal use in Finland.
Table 1. Age, sex and lactate transport activity based on the amount of CD147 in 3 horse breeds
HT = high lactate transport activity, LT = low lactate transport activity. Age is presented as median (interquartile range). *The percentage of HT horses in Thoroughbreds differed significantly (P<0.05) from that in Standardbreds.
Plasma membranes of RBC were isolated as described previously (Koho et al. 2002). In brief, cells were incubated in 5 mmol/l sodiumphosphate buffer at pH 8.0 to achieve complete haemolysis and membranes were washed 3 or 4 times with the same buffer to remove haemoglobin. The plasma membranes were separated with centrifugation in a Percoll gradient.
Information about horse MCT1 and CD147 sequences (GenBank accession no. AY457175.1; EF564280.1) were used to design antibodies. The C-terminus of horse MCT2 was sequenced and the antibody designed accordingly (the sequence corresponded to the predicted horse MCT2 sequence, GenBank accession no. XM_001490658). Antibodies against the C-terminus of horse MCT1 (CKGTEGDPKEESPL; GenBank accession no. AAR21622.1), MCT2 (CQSARTEDHPSERETNI; GenBank accession no. XP_001490708) and CD147 (CGHHVNDKDKNVRQRNAS; GenBank accession no. ABQ53583.1) were raised in rabbit and purified with affinity chromatography1. Membrane proteins (40 µg/lane) in 2 × Laemmli loading buffer were separated on 10% (w/v) SDS-PAGE gels and blotted onto nitrocellulose filters (Protran)2. Filters were stained with 0.5% Ponceau S in 1% acetic acid for 2 min, washed in water and visualised with AlphaImage3 to detect protein. Blots were blocked with 10% dry milk in Tris buffered saline with 0.1% Tween (TBST). Filters were incubated overnight at +4°C with primary antibodies diluted in blocking buffer and washed for 15 min + 2 × 5 min with TBST. Filters were then incubated with horseradish peroxidise-conjugated anti-rabbit antibody4 in TBST, supplemented with 2.5% dry milk for 1 h at room temperature. Washes were repeated as above. Visualisation was performed by adding chemiluminescence reagent (Supersignal West Dura)5 to filters. Filters were imaged with LAS-3000 luminescent image analyser6. Blots were quantified using AIDA image data analyser7. Each gel contained 2 control samples, one pooled from horses known to express low amounts of CD147 and the other from horses known to express high amounts of CD147 (Koho et al. 2002, 2006). The optical density of these controls was set to be 1 and 100, respectively. The optical densities of the samples are expressed in relation to these controls. The specificity of antibodies against MCT1, MCT2 and CD147 was tested by blocking the antibody reactivity with respective peptide (Fig 1).
Racing information of individual Thoroughbred horses was obtained from the archived data of the Racing Post8, which contains the race and performance history for racing Thoroughbreds involved in flat racing, National Hunt and point-to-point racing in the UK. Racing Post ratings (RPR), official ratings (OR), top speed (TS) and career prize money were used to evaluate racing performance. Official ratings are compiled by the British Horse Racing Board and are used to determine the weights horses will carry in handicap races. Top speed ratings are based on race times, with horses recording faster times achieving higher ratings.
Differences between groups were calculated using a Mann-Whitney test and a Chi-squared test. Correlations were calculated with Spearman's correlation. The bimodality of distributions was tested with an F test following curve fitting (Origin 7.5)9. Values of P<0.05 were considered significant.
Western blot analysis
Antibodies against horse CD147, MCT1 and MCT2 were used to detect isoform expression in RBC membranes. Based on the amount of CD147, the horses could be divided into two groups in all breeds. In the high lactate transport activity group (HT) horses CD147 was found at about 50 kDa bands. In the low lactate transport activity group (LT) horses the band at 50 kDa was very faint or absent (Fig 1). The intensity of the 50 kDa band was significantly higher (P<0.001) in the HT horses than in the LT horses in all breeds.
MCT1 was present on western blot as a double band at about 47 and 50 kDa. The MCT1 bands were faint or absent in horses in the LT group (Fig 1). The intensity of the MCT1 bands was significantly higher (P<0.001) in the HT horses than in the LT horses in all breeds. MCT2 was found at about 90 kDa band in both HT and LT horses (Fig 1). The amount of MCT1 correlated with the amount of CD147 in all breeds (P<0.001). There was no correlation between MCT2 and CD147 or MCT1. There was no correlation with age and the amount of CD147, MCT1 or MCT2.
The distribution of the amount of CD147 was clearly bimodal in FH and SB with 85 and 82% expressing high amount of CD147, respectively (Fig 2, Table 1). In TB, 88% had high expression of CD147 and 11% low expression. More horses belonged to the HT group in the TB compared to SB (Table 1). There was no difference in the amount of horses in the HT group in FH compared to SB or TB. One TB horse (1%) had intermediate expression of CD147 and could not be included into either group (Fig 2). Such an intermediate expression was not apparent in FH and SB. The amount of MCT1 followed the bimodal distribution of CD147 but was statistically significant only in the TB (Fig 2). The amount of MCT2 was distributed unimodally and the 2 groups could not be distinguished (Fig 2).
Finnhorse mares had more MCT2 (P<0.05) than colts and geldings. TB mares had more CD147 (P<0.05) than colts and geldings. When all breeds were combined, there were no differences between sexes.
Racing performance data were available for 77 of the Thoroughbred racehorses. Best RPR varied from 47–149 (median 89; IQR 110–70), best OR was 40–149 (median 87; IQR 108–70), Best TS varied from 16–137 (median 76; IQR 100–53) and career prize money was £0–414,872 (median £4,637; IQR £19,300–287). Colts and geldings had higher best RPR, best TS and best OR compared to mares. Performance markers did not correlate with the amount of MCT1, MCT2 or CD147 in TB RBC membranes.
In all breeds studied, the horses could be divided into 2 groups based on the amount of CD147 in RBC membranes (Table 1). The abundance of CD147 indicates high lactate transport activity (HT), while a low amount indicates low lactate transport activity (LT; Koho et al. 2002). In the Standardbred, we found that 82% of the horses studied belonged to the HT group. Our results are in accordance with an earlier study with 89 horses which reported 75% of Standardbreds to have high lactate transport activity (Väihkönen and Pösö 1998). The percentage of Thoroughbred racehorses in the HT group (88%) was greater when compared to Standardbreds. However, almost as many Finnhorses (85%) belonged to the HT group as Thoroughbreds. This was an unexpected finding, since the racing Thoroughbred, bred for centuries to race at high speeds generating blood lactate concentrations >25 mmol/l (Harris and Snow 1988), might be anticipated to have different lactate transport profile, compared to the Finnhorse, which has a shorter history as a racehorse. The Finnhorse has also been shown to have lower blood lactate concentrations than SBs after maximal exercise (Pösöet al. 1983).
One Thoroughbred racehorse showed intermediate expression of CD147 and therefore could not be grouped into either HT or LT group. Previously, one Standardbred racehorse, with similar medium lactate transport activity based on the amount of CD147, has been found (Koho et al. 2006). Since only 2 horses with intermediate CD147 expression have been reported to date, it is not possible to say whether they represent a third, rare group, or a methodological error.
Finnhorse mares had more MCT2 in their RBC membranes than stallions and TB mares had more CD147 in their RBC membranes than colts. Koho et al. (2002) have shown that the amount of CD147 correlates positively with lactate transport activity. Thus the sex difference found in TB is in agreement with the finding by Väihkönen and Pösö (1998) who showed that lactate transport activity in RBC of SB mares was higher than that of stallions.
In the horse, a vast majority of total lactate transport into RBCs has been shown to be due to MCTs (Skelton et al. 1995; Väihkönen and Pösö 1998). Up to 50% of blood lactate can be found in RBCs after intense exercise (Pösöet al. 1995; Väihkönen et al. 1999). In human athletes the percentage of lactate in RBC is around 20% (Juel et al. 1990; Smith et al. 1997). It has been speculated that the influx of lactate from plasma into RBC sustains the gradient between muscle cell and plasma enabling more lactate to be produced in the muscle cells (Pösöet al. 1995; Juel et al. 2003). This might be beneficial in high intensity exercise. In fact, Räsänen et al. (1995) found that horses with high amounts of lactate in their RBCs after a trotting race, had better performance indexes than the horses with low amounts of lactate in RBCs. However, in the present study, we could not show any correlation between racing success and the amount of lactate transporting proteins in TBs. This finding is similar to that of Väihkönen et al. (1999). The variation between results highlights the challenge of developing appropriate performance markers in horses as the performance markers all depend on age. In addition, the variability of performance results is further increased by Thoroughbred racehorses competing in several types of races, including steeple chase, hurdles and flat racing. The range of performance indexes in the horses studied by Räsänen et al. (1995) was large and the group included very poor performers and top athletes, whereas in the study by Väihkönen et al. (1999) where no correlation with the RBC lactate concentration and performance index was found, the range of the performance indexes was rather narrow. To demonstrate a correlation between lactate transport activity and performance, a larger number of horses with a large range of performance capacity, but the same age, needs to be studied.
The lactate transport activity of red blood cells in other athletic species (human, greyhound and reindeer) have been previously studied (Skelton et al. 1995; Väihkönen et al. 2001). Väihkönen et al. (2001) showed that the horse is the only species where lactate transport activity is bimodally distributed. In previous studies, the 2 groups could already be distinguished in foals and horses would remain in their group throughout later life (Väihkönen and Pösö 1998; Väihkönen et al. 1999). It has been suggested that in the Standardbred, low lactate transport activity is inherited as an autosomal recessive trait (Väihkönen et al. 2002). Previously, training has been shown to increase RBC lactate transport activity in reindeer and sled dogs, but not in horses (Väihkönen et al. 2001). In man, the effect of training is controversial, as both increase in RBC lactate transport activity and no effect have been previously reported (Skelton et al. 1998; Väihkönen et al. 2001). In the present study, age did not affect the amount of lactate transport proteins in any breed. While age would not be anticipated to affect genotype, it was assumed that older horses would have experienced a greater amount of training, and these findings support there also being no effect of training on lactate transport protein expression.
As discussed above, the connection between the expression of lactate transporters and racing performance is difficult to prove. As we show here, a substantial number of horses at least in 3 breeds show differences in the expression MCT1 and CD147 in their RBC membranes. In previous studies, the amount of MCT1 has not varied between the 2 groups (Koho et al. 2002). This has apparently been due to unspecific binding of the antibody, that has been designed against human MCT1. The discrepancies between our current and previous results highlight the fact that only antibodies validated to the species in question should be used. In the present study we measured the amounts of MCT1 and CD147 in the membranes where they are bound together and form the active transporter complex (Kirk et al. 2000). However, MCT1 and CD147 are already bound together on the endoplasmic reticulum and only the complex is translocated to the membranes (Gallagher et al. 2007). Therefore, the lack of MCT1 would result in low amount of CD147 in the membrane and vice versa. Accordingly, the low lactate transport activity could be due to lack of CD147 only.
While MCT1 is a monocarboxylate transporter, CD147 has also several other functions. It is widely expressed in the body and is known to interact with various proteins, in addition to MCTs (Iacono et al. 2007). Therefore, a complete inability to produce CD147 would have multiple severe consequences to the individual. This is evident as CD147 knockout mice are usually unable to undergo implantation and are small and sterile if they survive (Igakura et al. 1998).
To our knowledge, the horse is the only species where MCT2 has been found in RBC membranes (Koho et al. 2002, 2006). In this study, the expression of MCT2 varied between individuals, but there was no difference between HT and LT groups. Our finding is in accordance with earlier results (Koho et al. 2002, 2006). MCT2 needs another ancillary protein to function and therefore CD147 does not affect the transport activity of MCT2 (Wilson et al. 2005). The high molecular weight of MCT2 compared to MCT1 indicates that the protein is either in a dimeric form or attached to its ancillary protein in our western blots. According to Wilson et al. (2005), such a dimer of membrane proteins may be stable enough to withstand the denaturating conditions of SDS-PAGE. Previously, it has been suggested that MCT2 transports lactate at low concentrations in horses, indicating that MCT1 is more important during exercise (Koho et al. 2002).
In conclusion, we found that 2 groups of lactate transporter expression in RBC membranes were present in all 3 horse breeds studied. The greatest proportion of high lactate transport activity was found in the TB. Unexpectedly, a large number of FH were also found with high lactate transport activity. One TB had intermediate expression. There was no significant statistical correlation found between lactate transporters in RBC membrane and markers of racing performance in the Thoroughbred.
The authors wish to thank Jyrki Kukkonen for statistical advice and Ninna Koho for scientific assistance.
Conflicts of interest
The authors have declared no potential conflicts.
1 Sigma Genosys, Pampisford, Cambridgeshire, UK.
2 Schleicher & Schuell, Dassel, Germany.
3 Alpha Innotech, San Leandro, California, USA.
4 DAKO, Glostrup, Denmark.
5 Pierce, Rockford, Illinois, USA.
6 Fujifilm Life Science, Dusseldorf, Germany.
7 Raytest, Straubenhardt, Germany.
8 Racing Post, London, UK.
9 OriginLab Corporation, Northampton, Massachusetts, USA.