Genes responsible for adaptation to exercise, especially endurance exercise that is unique and requires the involvement of many processes, are still being searched for. Based on previous research in horses the candidate genes included the ACTN3 [13] and the KL [7]. Both genes were considered as involved in exercise effort in horses based on whole transcriptome sequencing of muscle or/and blood [13, 7]. Moreover, their role in metabolism regulation (ACTN3, KL), inhibition of oxidative stress, and controlling of Ca2+ and phosphate homeostasis [4] confirms the need to test both genes for their involvement in exercise-related responses in horses.
The ACTN3 gene encodes α-actinin-3, the protein specifically expressed in type II (fast-twitch) muscle fibers designed for quick, forceful contractions and are particularly prevalent in muscles involved in sprint and other power activities with very high energy consumption. On the contrary, the type I muscle fibers are characterised by slow twitch and are more efficient in aerobic effort. The study of Norman et al., (2014) [17] has shown that the lack of functional α-actinin-3 is associated with reduced activity of glycogen phosphorylase and increased activity of glycogen synthase, as well as higher muscle glycogen content. The authors suggested that these changes lead to a decreased capacity of muscle to use glycogen as a fuel and increased consumption of simple carbohydrates resulting in higher ATP production. That allows to conclude that the presence or absence of α-actinin-3 due to the variations in ACTN3 gene variants, along with its isoforms, significantly affects muscle function and athletic performance via metabolism modification. In human athletes, these findings allowed to use ACTN3 gene as a genetic marker of sprint or marathon effort predisposition [10].
Except from humans studies, very promising results have also been obtained in equines. The downregulation of ACTN3 gene was detected for the first time in Arabian horses during whole transcriptome analysis on muscle tissue during the training of young horses to flat-races [12]. The significance of ACTN3 extends into its isoforms that can result from gene expression modulation, alternative splicing, or post-translational modifications, each potentially having distinct roles in muscle physiology. The decreased expression of both isoforms and total variant of the ACTN3 gene after the endurance ride is in accordance with the downregulation described previously in Arabian horses that took part in flat racing [13].
The results o this study, conducted on Arabian horses competed in the 120 km ride confirmed that the downregulation of the v202 isoform showed the highest rate – 2.75 fold, while the share of that isoform was the lowest. Such results may suggest that the protein encoded by the v202 variant has a limited role in muscle metabolism during endurance effort, much less than other α-actinin-3 isoforms. In comparison, the v201 downregulation rate was 1.80-fold and the expression level was higher than v202 isoform in every case, but still represented a small share of the total variant. In the study of Almarzook et al., (2019) [18] the genetic variation of ACTN3 was investigated in terms of endurance exercise in Arabian horses. The authors suggested that the occurrence of a SNP 12:26524930T > C mutation in 3’UTR may significantly affect gene expression, and consequently, result in better predispositions for endurance rides. What is also significant, the recent studies on Arabian and Thoroughbred horses have shown a very high degree of variation in the ACTN3 gene in response to various conditions like breeding strategies and selection pressure [19].
It's worth to mention that molecular network of ACTN3 gene showed the direct association with SH3RF2 gene, described as cell anti-apoptotic factor. The previous studies have shown a significant downregulation of SH3RF2 during intensive physical effort, concluding that apoptosis leads to replacing damaged muscle fibers by new, better suited to exercise [15]. As both SH3RF2 and ACTN3 genes could be potentially used as genetic markers of endurance predispositions in horses, their influence at the molecular level can be assumed and may constitute the basis for further research. Moreover, the obtained association network have presented the MIOX gene as a molecular link between other analysed genes. The MIOX gene encodes the myo-inositol oxygenase, first and rate-limiting enzyme in myo-inositol (MI) metabolism pathway and has not been investigated to date in horse. The increase in MIOX enzyme activity is in proportion to serum glucose concentrations and plays an important role in carbohydrate metabolism [20]. That finding provides a new look at the MIOX gene and creates opportunities for further research in the context of understanding the endurance predispositions of horses.
The KL gene encodes klotho protein, a transmembrane β-glucuronidase (EC number 3.2.1.31) capable of hydrolysing steroid β-glucuronides, described for the first time in 1997 by Makoto Kuro-o et al. [21] The primary function of klotho is to activate the ion channel TRPV5, providing control over the sensitivity of the organism to insulin. Moreover, it influences intracellular signalling pathways including p53/p21, cAMP, protein kinase C (PKC) and WNT signalling pathways modulating Ca2+ and phosphate homeostasis [22, 4]. These features have encouraged further studies and analyses. about the role of KL in sports. In the study of Kim et al., (2015) [23] the klotho was described as an ageing suppressor, being a crucial molecule in aging processes and its overexpression resulted in longevity. In turn, the research performed on KL deficient mouse model in endurance running strongly indicated that this factor can be a key modulator of muscle performance [5].
In the horses, the KL gene was described by [7] where its significant downregulation in response to intense physical effort was depicted using the high-throughput RNA-seq screening method. The results indicated that the expression level of KL gene can be considered as exhaustion biomarker, and a lower expression would be indicative of a better endurance ability in equines.
The results obtained within the present study have confirmed the downregulation of the KL gene as well as its two isoforms – v202 and v203. The significant differences in the expression level before and after the 120 km ride were obtained for the total KL expression and v202 variant. Interestingly, the transcript abundance of the v203 isoform was the highest in every conditions in comparison to the others – the v203 isoform compared to v202 in both measurement time – before and after the ride showed 45.5 – fold and 96 fold ; respectively. While the downregulation of KL in terms of endurance exercise is confirmed and considered as preferred and beneficial, the expression of the v203 variant was relatively high. This fact may suggest another role of the klotho protein encoded by v203 variant in endurance effort that has not been described so far. While the recent studies have investigated and described the KL expression and role in mice model [24] and in humans [25], the results obtained in the present study, on the basis of field observations demonstrated the possibility of carrying out further research on equines.
The endurance exercise is a complex issue considering the complexity of biochemical changes occurring in the organism. In the present study we have shown that both ACTN3 and KL genes come in different variants which expression levels differs between each other. Our results also suggest a significant interaction between particular group of genes in terms of adaptation to physical exertion, where the SH3RF2 and MIOX genes play a crucial role. Further research in that matter may result in a wider knowledge of that topic and in describing genetic markers predisposing to a long-term exercise.
The main limitation of the study were the small research group and the homogeneity of the group (different age, differences in the conditions of transport to the competition venue). These limitations cannot be avoided in the field studies, which on the other hand pose the unique possibility to examine sport horses in the reality of competitions.