Biological activity of IL-15 in vivo is likely regulated through interactions with IL-15Rα, which may represent an important mechanism for controlling overall IL-15/IL-15Rα signaling and/or biological activity. This study provides physiological and molecular data demonstrating that loss of IL-15Rα in vivo results in remodeling of fast muscles to a slower, more oxidative phenotype, akin to the roles proposed for PGC-1α and ACTN3
). These observations were specific to the loss of IL-15Rα, as knockout of IL-15 and transgenic overexpression of IL-15 did not result in altered exercise and muscle phenotypes. If interactions between IL-15 and IL-15Rα were to occur strictly as a secreted cytokine binding to a cell surface receptor, then similar exercise and muscle phenotypes would have been observed in both IL-15Rα–KO mice and IL-15–KO mice, since ligand-receptor binding would have been interrupted in both of these mouse strains. Because the phenotypes were distinct in these knockout mouse models with respect to spontaneous cage activity and fast muscle isometric contractile characteristics, we can conclude that the roles of IL-15Rα and IL-15 in vivo are distinct with regard to muscle function. On this basis, we propose that IL-15Rα has a role in determining the contractile and morphological phenotypes of fast skeletal muscles in vivo. This hypothesis is supported by the allelic associations between a SNP in the human IL15RA
gene and endurance athlete status.
Adding to the documented complexity of IL-15–related activity, multiple isoforms and splice variants of IL-15Rα exist as both membrane-bound and soluble forms that can either inhibit or potentiate IL-15 biological activity (29
). The current study demonstrates that complete knockout of IL-15Rα results in a mouse with an increased capacity for activity and initiates a remodeling of fast skeletal muscles to a more oxidative phenotype, phenotypes that were specific to knockout of IL-15Rα. It is also interesting to note that deletion of IL-15 and overexpression of IL-15 did not result in opposite muscle physiological responses, suggesting that interaction of IL-15Rα with IL-15 represents a mechanism by which biological activity of IL-15 is controlled. Indeed, the observation that knockout or transgenic overexpression of IL-15 did not alter exercise or muscle phenotypes strengthens this possibility. Furthermore, previous reports have not been able to demonstrate consistently that increasing IL-15 levels in vivo exerts a significant therapeutic effect in skeletal muscle (11
). However, reductions in soluble IL-15Rα in aged muscle were correlated with reductions in circulating IL-15 levels (6
). These data clearly demonstrate that the relationship between IL-15 and IL-15Rα is more complex than a ligand–membrane-bound receptor interaction, highlighting a unique and, to our knowledge, previously undefined role for IL-15Rα in muscle physiology and morphology.
Several physiologic parameters were measured to characterize exercise and muscle properties in these mouse strains, and three key observations support our hypothesis of a role for IL-15Rα in fast muscle phenotypes: (a) fast muscles with an increased resistance to fatigue; (b) fast muscles with isometric contractile properties more similar to those of slow muscles; and (c) greater total running activity in IL-15Rα–KO mice. Resistance to repeated contraction-induced fatigue is a hallmark characteristic of slow skeletal muscles such as the soleus and is due to a combination of increased mitochondrial volume, oxidative enzyme capacity, capillarity, and smaller muscle fiber sizes. In contrast, the fast EDL muscle fatigues quickly due to a lower mitochondrial volume, increased glycolytic enzyme capacity, reduced capillarity, and larger muscle fiber sizes (42
). EDL muscle from IL-15Rα–KO mice displayed an increase in fatigue resistance greater than control EDL muscles, although this was not a full transition to a slow phenotype. Specifically, the fatigue index curve of the EDL from IL-15Rα–KO mice was indistinguishable from the fatigue index curve of a soleus muscle from a control mouse during the first 30–40 seconds of the repeated stimulation protocol. Also, compared with an EDL muscle from a control mouse, the fatigue curve was shifted entirely to the right during the first 70 seconds of the repeated stimulation protocol, after which the fatigue curves of EDL muscles from B6129 control and IL-15Rα–KO mice were not different. Previous studies have reported an increase in fatigue resistance in EDL muscles following genetic manipulation of Actn3
) and Pgc-1
), although differing fatigue protocols preclude direct comparison with the current data in the IL-15Rα–KO mouse.
The isometric contractile properties of EDL muscles from IL-15Rα–KO mice were also consistent with the adoption of a slower contractile phenotype. Force production in response to twitch and tetanic muscle contractions resulted in lower forces in EDL muscles from IL-15Rα–KO mice compared with B6129 control mice, and were visibly lower than forces from wild-type C57BL/6 mice. This was true whether force was expressed in absolute terms or normalized to muscle CSA, indicating a functional alteration of the muscle independent of size. Interestingly, the twitch/tetanus ratio in EDL muscles from IL-15Rα–KO mice was 15% lower than in EDL muscles from control mice. The lower value for the twitch/tetanus ratio observed in this study is consistent with a previous report noting lower twitch/tetanus ratio values in muscles composed of slower motor units (43
), and similar findings have been previously reported in the Actn3
-KO mouse (40
). In addition, the altered rates of force development and relaxation suggest alterations in the regulation of intracellular calcium levels following maximal contractions, as are noted to occur in slow skeletal muscles (44
). Last, spontaneous cage activity was more than 6-fold greater in IL-15Rα–KO mice compared with B6129 control mice, and this amount of cage running activity was greater than that noted in other mouse models, including the HIF-1α–KO mouse (45
) and mice treated with PPAR agonists (46
). Collectively, the muscle contractile and spontaneous running experiments clearly demonstrate a more oxidative phenotype that is specific to the loss of IL-15Rα.
Quantification of several parameters confirmed altered morphology in fast skeletal muscles deficient in IL-15Rα, and the data support the hypothesis that loss of IL-15Rα promotes a slower, more oxidative muscle phenotype. Slow muscles that are tonically active (e.g., soleus muscle) are composed of muscle fibers with smaller CSAs (42
). The single-fiber area histograms for both the EDL and TA muscles from IL-15Rα–KO mice were shifted to the left, indicating smaller fiber sizes. Interestingly, the single-fiber area histogram of soleus muscles from IL-15Rα–KO mice was not different from control, demonstrating a specific effect of IL-15Rα knockout on fast muscle morphology. In addition, the total fiber number and the percentage of CNFs were greater in fast muscles from IL-15Rα–KO mice and were not different from control in the slow soleus muscle. Previous studies detailing mouse models of increased endurance capacity and oxidative potential, including PGC-1α transgenic (41
), HIF-1α–KO (45
), and Actn3
-KO mice (40
), did not report changes in muscle fiber number. The ratio of nuclei per average single-fiber area was also larger in EDL and TA muscles from IL-15Rα–KO mice, an observation previously noted in slower skeletal muscle (37
). These morphological data highlight a unique feature of fast muscles that are IL-15Rα deficient, which likely contributes to the slower contractile characteristics we observed in these normally fast muscles.
The molecular signature of muscles from IL-15Rα–KO mice provides a plausible mechanism for the altered exercise and muscle physiological properties we observed. Indeed, an analogy can be drawn to the extraocular muscles, which can contract rapidly for long periods of time and have a distinct molecular makeup compared with skeletal muscles (47
). The increased resistance to fatigue of the fast EDL muscle in response to repeated stimulation suggested an alteration in mitochondrial content and/or function. The observation that SDH activity was greater in EDL muscles from IL-15Rα–KO mice supports the physiological demonstration of a greater resistance to fatigue in these fast muscles. In addition, knockout of IL-15Rα results in an upregulation of PPARδ and PGC-1α, along with changes in mitochondrial enzymatic activity and oxidative potential. The transcription factor PPARδ and the coactivator PGC-1α are important regulators of oxidative and mitochondrial genes. For example, slow muscles have greater amounts of PGC-1α (41
), and overexpression of PGC-1α drives mitochondrial biogenesis and promotes an increase in oxidative potential, while PGC-1α–knockout mice have a reduced endurance capacity along with increased muscle fiber damage and inflammation following exercise (41
). The observations of altered rates of force development and relaxation during maximal twitch contractions suggested an alteration in calcium-related markers in the muscles from IL-15Rα–KO mice. Calcium kinetics as well as calcium-handling proteins differ between fast and slow muscles. The muscles from IL-15Rα–KO mice had a greater relative expression of the SERCAII isoform and lower mRNA and protein levels of calsequestrin, both characteristic of slow muscles (52
). The observation of reduced calsequestrin in the muscles of IL-15Rα–KO mice is interesting inasmuch as calsequestrin concentrations are reported to be higher in fast muscles (52
) and fast muscles from calsequestrin-1–null mice display contractile characteristics similar to those of slow muscles, including longer contraction times and half-relaxation times (ν RTs) (53
). These data demonstrate an altered molecular state of skeletal muscle deficient in IL-15Rα that contributes to the changes noted in muscle function and cage activity.
IL-15Rα–KO mice weighed almost 20% less than B6129 control mice, and this was reflected in the lower absolute weights of individual skeletal muscles. However, when muscle weights were normalized to lean muscle mass, the muscle/lean muscle mass ratios were greater in IL-15Rα–KO mice. This is interesting, given that muscle/lean muscle mass ratios of non-skeletal muscle organs from IL-15Rα–KO mice, such as the heart, spleen, and kidneys, were not significantly different from those of B6129 controls. These data demonstrate a preferential effect of loss of IL-15Rα on normalized skeletal muscle weight, which may reflect alterations in overall body metabolism, as previously suggested (14
). He et al. (14
) measured horizontal and vertical beam breaks in a metabolic chamber and reported a significant increase in activity levels of female IL-15Rα–KO compared with control mice. Similarly, we observed that male IL-15Rα–KO mice produced 6.3-fold more wheel revolutions compared with B1629 control mice in cage wheel running experiments and had a larger number of ambulatory photobeam breaks in the light and dark cycles. Although the cage running wheel system and the ambulatory photobeam break system used in the current study are measures of spontaneous activity, the dramatic increases observed in both studies reflect a consistent behavior of increased activity that suggests an increase in the capacity to perform exercise. He et al. (14
) also report an increased VO2
in these mice, which, along with a leaner body composition, would suggest an altered metabolic phenotype, supporting the data in the current study. The upregulation of Il15
mRNA in skeletal muscles from IL-15Rα–KO mice reported in the current study could potentially contribute to the altered body composition observed in these mice (14
), as HSA-IL-15TG mice had lower adiposity and were resistant to high-fat diet–induced obesity (34
The contribution of central versus peripheral influences to the observed phenotype in the IL-15Rα–KO mouse has not been clarified. Both IL-15Rα and IL-15 are expressed in the central nervous system, and circadian sleep patterns are altered with manipulation of these molecules, demonstrating an effect in these tissues (54
). Recent studies have also suggested a role for IL-15Rα in altering circadian rhythms of activity and thermoregulation (14
), as well as in normal anxiety behavior (15
). Although our data in the IL-15Rα–KO mouse do not provide an explanation for the influence of central versus peripheral mechanisms on the observed exercise and muscle contractile phenotypes, the ex vivo muscle physiological data demonstrate that the end result is altered fast muscle morphology and contractile characteristics. Since levels of PGC-1α and citrate synthase were unchanged in spleen and kidney, in contrast to the situation in skeletal muscle, it would suggest that the mechanism of the increased endurance and altered metabolic characteristics is a muscle-specific adaptation to the lack of IL-15Rα. Experiments are being planned to delineate the central and peripheral mechanisms underlying the observed phenotype in these mice.
SNPs can modulate gene function and/or expression, and SNP association studies can provide preliminary data for further hypothesis-directed experiments. SNPs in the human IL15
genes have been associated with responses of skeletal muscle to resistance training (17
), baseline measures of skeletal muscle and bone (16
), and markers of the metabolic syndrome (16
), suggesting a role for these genes in skeletal muscle. Numerous reported SNPs in the IL15RA
gene occur at intron-exon borders, raising the possibility that these SNPs could affect expression levels, gene splicing, and/or processing (16
). In the current study, the genotype and allele frequency of a SNP in exon 3 of the IL15RA
gene were associated with elite human endurance athletes stratified by sport. However, this was an exploratory analysis, and additional SNPs in the IL15
genes and additional cohorts of athletes should be screened to further define the association between genotype and certain types of athletic performance; these analyses are currently being pursued. These data are compelling, given the increased activity and metabolic characteristics of IL-15Rα–KO mice observed in the current study.
In conclusion, our results support the hypothesis that IL-15Rα has a role in defining the phenotype of fast skeletal muscles in vivo. Physiological evaluation of IL-15Rα–KO mice demonstrated increased exercise capacity and altered muscle contractile properties consistent with a transition of fast muscles toward a slower contractile phenotype. These altered physiological parameters may be explained by alterations in markers involved in mitochondrial biogenesis and calcium kinetics. Positive genetic associations (genotype, allele frequency) were found between a SNP in the IL15RA
gene and endurance in athletes stratified by sport, suggesting a conserved role of IL-15Rα and endurance. Although the current study demonstrates that loss of IL-15Rα promotes this phenotype, future studies are needed to determine the roles of specific IL-15Rα isoforms in muscle, as well as the associations of additional IL15RA
polymorphisms in muscle phenotypes in humans. Our study highlights a heretofore unrecognized role for IL-15Rα in muscle and the unique muscle and exercise phenotypes caused by IL-15Rα deletion. We suggest these observations will have therapeutic implications for skeletal myopathies and systemic diseases such as diabetes and obesity and for the aged population, in which reduced endurance impacts mortality and activities of daily living (57
); as well as societal implications in terms of altering athletic performance.