Systemic OT and OTR levels in satellite cells decline with age

Since circulating OT levels decrease after ovariectomy, which mimics hormonal ageing11, we hypothesized that its level would decrease during natural ageing. OT plasma levels were measured in young (2- to 4-month-old) and old (18- to 24-month-old) C57/BL6 male mice, using an OT-specific enzyme immunoassay. A significant threefold decrease was observed in aged mice as compared with young, suggesting that endocrine levels of OT decline with age (Fig. 1a). Little is known about the expression of the OTR in skeletal muscle either in general or in an age-specific way; thus, we examined OTR protein expression in young and old skeletal muscle. Western blotting analysis demonstrates that OTR is detected in whole skeletal muscle (Fig. 1b). Similar levels of OTR were observed in young and old whole muscle lysates that contain, in addition to proteins from muscle fibres and satellite cells, proteins from other cell types including vascular and immune cells, adipocytes and fibroblasts (Fig. 1b,d). Notably, when protein lysates were prepared from muscle stem cells, OTR was found to be expressed at significantly higher levels in young satellite cells as compared with old (Fig. 1c,e). These results demonstrate that with ageing the decline in the circulating OT hormone is compounded by diminished levels of OTR in the old muscle stem cells. Confirming the western blotting data, immunofluorescence analysis of muscle tissue sections revealed that OTR was indeed expressed in cells that were observed in the satellite cell position (Fig. 1f, top) and colocalized with the satellite cell marker Pax7 in quiescent and injury-activated satellite cells (Fig. 1f, middle panels). Although most Pax7-positive satellite cells express OTR, expression of this receptor was also observed in many Pax7-negative cells (Fig. 1f), suggesting that OT acts on several cell types in skeletal muscle.

Figure 1: Systemic oxytocin declines with age and oxytocin receptor is expressed in skeletal muscle satellite cells. (a) OT plasmatic levels were quantified using enzyme immunoassay in young (2- to 4-month-old) and aged (18- to 24-month-old) C57BL/6 male mice. Data represent mean±s.e.m. (n=6 young versus n=7 old). Two-tailed unpaired Student’s t-test, **P value <0.01. (b,c) Whole skeletal muscle (gastrocnemius) protein extracts (b), and hind limb satellite cell protein extracts (c) were prepared from young and old non-injured mice. OTR, Pax7 and myosin heavy chain (MHC) were assayed using western blot analysis. Thirty micrograms of protein were loaded per lane and β-actin was used as a loading control. In b,c, Y-SKM: young skeletal muscle; O-SKM: old skeletal muscle; Y-SC: young satellite cell; O-SC: old satellite cell. (d,e) Quantification of western blot from b,c, using the Image J software. Data represent mean±s.e.m. (n=4 Y-SKM, n=4 O-SKM, n=3 Y-SC and n=3 O-SC). Two-tailed unpaired Student’s t-test, *P value <0.05. (f) Cross-sections from non-injured and 3-day-injured tibialis anterior muscle were immunostained for OTR and dystrophin or OTR and Pax7, as indicated, and counterstained with DAPI. IgG control are displayed (bottom) and dashed lines delineate muscle fibres. Scale bars represent 20 μm. Full size image

Systemic decline in OT causes poor regeneration of old muscle

To determine whether the age-specific decrease in systemic OT is responsible for the decline in muscle regeneration that is typical of old mice, we systemically administered OT to old mice and an OT-selective antagonist (OTA) to young mice and studied the success in muscle regeneration after cardiotoxin-induced injury. The schematic for this study is shown in Fig. 2a. Five days post injury, robust muscle regeneration was observed in young mice, based on the high numbers of newly formed (eMyHC+) fibres with centrally located nuclei in the injured area (Fig. 2b,c). As expected, a significant decline in the formation of new muscle fibres was evident in old mice. Interestingly, subcutaneous injections of OT improved muscle regeneration in the old mice to a level comparable to the young. In young mice, in which muscle regeneration occurs efficiently, ectopic OT had no effect (Fig. 2b,c). Importantly, a significant decrease in muscle regeneration, to a level similar to that seen in old mice, ensued when young mice were administered with OTA (Fig. 2b,c).

Figure 2: Age-specific systemic oxytocin decline plays a key role in the defective muscle regeneration observed upon ageing. (a) Schematic of the experimental procedure. Cardiotoxin (CTX) muscle injury was performed on day 0. Four days before muscle injury and over the course of the experiment, mice were administered OT (1 μg g−1 of mice), OTA (2 μg g−1 of mice) or vehicle (HBSS) daily. Five days after muscle injury, mice were euthanized and gastrocnemius (GA) muscles were dissected. Cross-sections (10 μm) were stained for haematoxylin and eosin (H&E). (b) H&E (top) and eMyHC (bottom) staining of cardiotoxin-injured GA muscle cross-sections from mice injected with OT, OTA or vehicle (HBSS). Scale bars represent 50 μm. (c) Muscle regeneration was quantified by scoring the number of newly formed fibres (eMyHC-positive fibres with centrally located nuclei) in the injured area of GA cross-sections. Data represent mean±s.e.m. (n=9 YV, n=5 OV, n=6 YOT, n=6 OOT, n=5 YOTA). One-way analysis of variance (ANOVA) with post hoc Newman–Keuls test, **P value <0.01, ***P value <0.001, NS: not significant. (d) Fibrosis quantification of GA muscle cross-sections 5 days after injury. The fibrotic index represents the percentage of the injury area occupied by connective tissue. Data represent mean±s.e.m. (n=3 YV, n=3 OV, n=3 YOT, n=3 OOT, n=3 YOTA). One-way ANOVA with post hoc Newman–Keuls test, *P value <0.05, **P value <0.01, NS: not significant. In b–d, YOTA: young injected with OTA; YOT: young injected with OT; YV: young injected with vehicle, OV: old injected with vehicle; OOT: old injected with OT. Full size image

The deficiency in skeletal muscle regeneration observed in aged mice is associated with an increase in fibrotic tissue formation5,23. As expected, the fibrotic index was greater in old mice as compared with young (Fig. 2d and Supplementary Fig. 1a). Ectopic OT significantly decreased the fibrotic index in old mice, whereas OTA injections increased fibrosis in young mice (Fig. 2d and Supplementary Fig. 1a).

These data demonstrate that one consequence of the age-specific systemic decline in OT is poor regeneration of skeletal muscle accompanied by increased fibrosis. Importantly, ectopic OT rapidly rescued repair of damaged old muscle, while OTA quickly incapacitated the regeneration of young muscle.

OT mediates the regenerative potential of muscle stem cells

Satellite cell activation/proliferation are the main limiting steps in old muscle regeneration. Exposure to a young environment has been shown to restore muscle regeneration by promoting satellite cell proliferation3,4,24. Since ectopic OT exposure over a short period of time rescued the repair of damaged old muscle, we evaluated whether the decreased level of OT in old was responsible for the lack of satellite cell proliferation in vivo. OT was administered to old mice daily starting 6 days before cardiotoxin-induced muscle injury (Fig. 3a). To monitor cell division in vivo, BrdU was injected subcutaneously 12 h before mice were euthanized. Three days after injury, tibialis anterior (TA) muscles were isolated, and the percentages of BrdU and Desmin double-positive cells (that is, proliferating myogenic progenitor cells that were generated by satellite cells activated in response to tissue injury) were quantified in the injury area in muscle sections (Fig. 3b,c). As we have previously published, these myogenic cells co-express Pax7 and Desmin, while the non-myogenic Desmin-positive cells represent less than 2% in such experiments25,26. A threefold decrease in myogenic cell proliferation was observed in old muscles as compared with young (Fig. 3c). Importantly and in agreement with the rescue of muscle repair, ectopic OT significantly improved myogenic cell proliferation in vivo (Fig. 3c).

Figure 3: Oxytocin improves muscle stem cell proliferation in vivo after injury. (a) Schematic of the experimental procedure. Cardiotoxin (CTX) muscle injury was performed on day 0. Six days before muscle injury and over the course of the experiment, mice were administered OT (1 μg g−1 of mice) or vehicle (HBSS) daily. Twelve hours before they were euthanized, mice were administered with BrdU (50 μg g−1 of mice, IP). Three days after muscle injury, TA muscles were isolated. (b) Representative micrographs of 3-day-injured TA muscle cross-sections (10 μm) immunostained for BrdU and Desmin and counterstained with DAPI. Right panels represent magnifications of the dashed area from left panels. White arrow indicates a BrdU and Desmin double-positive cell, yellow and white arrowheads indicate BrdU or Desmin single-positive cells, respectively. Scale bars represent 100 μm (left panels) and 50 μm (right panels). (c) Quantification of the percentage of proliferating myogenic cells (Desmin+ and BrdU+). Data represent mean±s.e.m. (n=4 YV, n=4 OV, n=4 OOT), one-way ANOVA with post hoc Newman–Keuls test, *P value <0.05, **P value <0.01. Full size image

To further study the effect of OT on satellite cell proliferation and differentiation, we performed an ex vivo analysis, as described in the schematic of Fig. 4a. Three days post injury, muscles were isolated and digested into bulk myofibres as previously described25,27 and cells were cultured in their own mouse’s respective serum for 24 h (Fig. 4a). In order to label the cells that were activated in vivo in response to muscle injury, BrdU was injected subcutaneously 12 h before mice were euthanized. Myogenic proliferation of cells activated in vivo was quantified by calculating the percentage of BrdU and Desmin double-positive cells, which as mentioned above is a sensitive and accurate way to measure the age-specific differences in myogenicity24,25,27. A 41% decrease in proliferation was observed in the old activated satellite cells as compared with young (Fig. 4b, c). Confirming the in vivo data observed in Fig. 3c, ectopic OT restored the myogenic proliferation of old satellite cells to a level comparable to young. Moreover, decreased myogenic proliferation was observed when young mice were treated with OTA (Fig. 4b,c). We also observed that the proliferation of activated satellite cells derived from young mice administered with OT tended to increase.

Figure 4: Oxytocin rejuvenates muscle stem cell function in injured tissue. (a) Schematic of the experimental procedure. Cardiotoxin (CTX) muscle injury was performed on day 0. Six days before muscle injury and over the course of the experiment, mice were administered OT (1 μg g−1 of mice), OTA (2 μg g−1 of mice) or vehicle (HBSS) daily. Twelve hours before they were euthanized, mice were administered with BrdU (50 μg g−1 of mice, IP). Three days after muscle injury, gastrocnemius (GA) and tibialis anterior (TA) muscles were isolated and digested into bulk fibres. Cells were cultured in their mouse’s respective sera (shortened as ‘own’ on the schematic) for 24 h and either fixed and stained for Desmin and BrdU or induced to differentiate for 48 h and subsequently fixed and stained for eMyHC. (b,c) Myogenic cell proliferation. Muscle fibre-associated activated satellite cells were isolated 3 days after injury from mice injected with OT, OTA or vehicle (HBSS), plated in media containing their mouse’s respective sera and fixed 24 h after plating. (b) Representative micrographs of cells immunostained for Desmin and BrdU and counterstained with DAPI. Scale bars represent 50 μm. (c) Quantification of the percentage of proliferating myogenic cells (Desmin+ and BrdU+). Data represent mean±s.e.m. (n=6 YV, n=3 OV, n=3 YOT, n=3 OOT, n=5 YOTA). (d,e) Myogenic fusion index. Muscle fibre-associated activated satellite cells were isolated 3 days after injury from mice injected with OT, OTA or vehicle (HBSS) and plated in media containing their mouse’s respective sera for 24 h. Cells were then induced to differentiate in mitogen-low fusion medium for 48 h, fixed and immunostained for eMyHC using DAPI to label all nuclei. (d) Representative micrographs. Scale bar represents 50 μm. (e) Quantification of the percentage of nuclei in eMyHC+ myotubes. Data represent mean±s.e.m. (n=8 YV, n=5 OV, n=6 YOT, n=6 OOT, n=5 YOTA). In b–e YOTA: young injected with OTA; YOT: young injected with OT; YV: young injected with vehicle, OV: old injected with vehicle; OOT: old injected with OT. One-way ANOVA with post hoc Newman–Keuls test, *P value<0.05, **P value<0.01, ***P value<0.001, NS: not significant. Full size image

To study whether these Brdu and Desmin double-positive satellite cell progeny were functionally competent, we evaluated their myogenic differentiation potential. As illustrated in Fig. 4a, cells isolated from muscles at 3 days post injury were cultured for 24 h in their mouse’s respective serum and were switched to differentiation medium for 48 h, when a pronounced age-specific decline in generation of de novo myotubes is typically detected. The myogenic fusion index (the number of nuclei included in de novo formed eMyHC+ myotubes as a fraction of the total nuclei) was then scored to assess cell differentiation. Supporting the observed increase in the number of newly regenerated eMyHC+ fibres in vivo after OT injection (Fig. 2b,c), systemic administration of OT to old mice rejuvenated the myofibre-forming capacity of satellite cells responding to tissue injury (Fig. 4d,e). In agreement with the idea that OT is required for myogenicity, when OT signalling was antagonized in young mice not only the proliferation but also the differentiation declined to levels similar to those of old animals, with their low circulatory OT (Fig. 4d,e). These results demonstrate that myogenic proliferation and subsequent differentiation depend on ‘youthful’ levels of OT.

Since we showed that OTR was expressed in skeletal muscle satellite cells (Fig. 1c,e,f), we hypothesized that OT acts directly on myogenic cells. To test this hypothesis, satellite cells were freshly isolated from cardiotoxin-injured muscles and plated in medium containing their own mouse serum supplemented or not with OT for 24 h. As previously published28 and shown in Supplementary Fig. 2, satellite cell purity was similar for young and old cells and greater than 90%. OT increased the proliferative capacity of old satellite cells cultured in old serum to similar levels of young cells cultured in young serum, as shown by the increased percentage of Ki67-positive activated muscle stem cells and the diminished expression of the cyclin-dependent kinase inhibitor 1, p21 (Fig. 5a–c). No effects of OT were observed on young satellite cells, in agreement with the higher levels of endogenous OT in young circulation (Fig. 1a). OT also promoted the proliferation of primary myogenic progenitors as shown by a threefold increase in the percentage of BrdU-positive cells (Fig. 5d, e).

Figure 5: Oxytocin improves myogenic progenitor cell proliferation via activation of the MAPK/ERK pathway. (a,b) Activated satellite cells were isolated 3 days after injury from young or old mice, cultured for 24 h in media containing their own mouse’s respective sera supplemented with OT (30 nM), PD98059 MEK inhibitor (50 μM) or OT plus MEK inhibitor, fixed and stained for Ki67 and counterstained for DAPI. (a) Representative micrographs. Scale bars represent 200 μm. (b) Quantification of the percentage of proliferating (Ki67+) satellite cells. Data represent mean±s.e.m. (n=4 mice per group). Two-way ANOVA with post hoc Bonferroni test, **P value <0.01, ***P value <0.001, NS: not significant. (c) p21 mRNA relative expression analysed with qRT–PCR. RNA was extracted from satellite cells isolated and cultured as in a. GAPDH was used as reference gene (n=3 mice per group). Two-way ANOVA with post hoc Bonferroni test, **P value <0.01, ***P value <0.001, NS: not significant. (d,e) Primary myogenic progenitors were cultured in the presence or absence of OT (30 nM), and UO126 MEK inhibitor (10 μM) for 48 h. BrdU was added to the culture medium during the last hour. Cells were then fixed, immunostained for BrdU and counterstained with DAPI. (d) Representative micrographs. Scale bars represent 50 μm. (e) The percentages of BrdU-positive cells were scored. Data represent mean±s.e.m. (n=8 independent experiments performed on different primary cultures). One-way ANOVA with post hoc Newman–Keuls test, ***P value <0.001, NS: not significant. (f) Primary myogenic progenitors were serum-starved overnight and then treated with OT (30 nM) or OT (30 nM) plus UO126 MEK inhibitor (10 μM) for up to 20 min. ERK1/2 and phospho-ERK1/2 were assayed with western blot analysis. O: OT, OM: OT plus UO126 MEK inhibitor, UN: untreated. Full size image

OT directly acts in satellite cells via the MAPK/ERK pathway

Since the mitogen-activated protein kinase (MAPK) signalling pathway plays an important role in muscle stem cell activation/proliferation29,30,31,32,33,34 and phospho-ERK1/2 is a well-documented downstream effector of OT in other cell types11,35, we postulated that OT could be a physiological inducer of MAPK that promotes myogenic cell proliferation via the phosphorylation of ERK1/2. Indeed, addition of a MEK inhibitor (MEKi) decreased the proliferation of young satellite cells and increased the expression of p21 to old levels (Fig. 5a–c). In accordance with the idea that OT signals via MAPK in supporting adult myogenesis, the effects of ectopic OT on freshly isolated activated satellite cells and on primary myogenic progenitor cells were abolished in the presence of MEKi (Fig. 5a–e).

To confirm the ability of OT to induce ERK1/2 phosphorylation, primary myogenic progenitor cells were stimulated for 5–20 min with OT in the presence or absence of MEKi. ERK1/2 phosphorylation was greatly induced 5 min after stimulation with OT (Fig. 5f). At 10 min ERK1/2 phosphorylation started to decrease and returned to the basal level by 20 min (Fig. 5f). In support of functional effects of OT on myogenic cell proliferation shown above, MEKi prevented OT-induced ERK1/2 phosphorylation, reducing the levels of phospho-ERK1/2 below the basal level observed in untreated cells (Fig. 5f), all confirming activation of the MAPK pathway by its endogenous ligand for productive myogenic responses.

Ot-deficient mice display premature muscle ageing

To confirm these findings in a genetic model, we studied muscle regeneration after cardiotoxin-induced injury in Ot knockout (KO) mice (B6;129S-Oxttm1Wsy/J). As compared with their wild-type (WT) littermates, we observed a progressive decline in muscle regeneration of Ot KO mice that was noticeable at 3 months of age and became pronounced and significant at 12 months of age (Fig. 6a,b). The decline in muscle regeneration observed between 1-year-old WT and Ot KO mice is comparable to the one measured between young (2- to 4-month-old) and old (18- to 24-month-old) WT C57/BL6 mice. In accordance with the data on the in vivo decline in the myogenic cell proliferation in old C57/BL6 mice, a decrease in activated satellite cell proliferation was observed in Ot KO mice when compared with WT littermates (Fig. 6c). A significant decline in muscle maintenance and repair is not expected and was not observed in WT mice at 12 months of age (Fig. 6b); thus, it is very interesting that the lack of OT prematurely ages the animals in this aspect.

Figure 6: Impaired muscle regeneration in mice lacking oxytocin. (a) H&E (top) and eMyHC (bottom) of cardiotoxin-injured gastrocnemius (GA) muscle cross-sections from 12-month-old WT and Ot KO mice. Scale bars represent 50 μm. (b) Muscle regeneration quantification of 3- and 12-month-old WT or Ot KO mice was performed by scoring the number of newly formed fibres (eMyHC-positive fibres with centrally located nuclei) in the injured area of GA cross-sections. Data represent mean±s.e.m. (n=6 WT and n=4 KO for the 3-month-old mice, n=4 WT and n=5 KO for the 12-month-old). Two-way ANOVA with post hoc Bonferroni test, *P value <0.05, NS: not significant. (c) Quantification of the percentage of proliferating myogenic cells (Desmin+ and BrdU+) of 3-day-injured TA muscle cross-sections immunostained for BrdU and Desmin. Data represent mean±s.e.m. (n=5 WT, n=5 KO). Two-tailed unpaired Student’s t-test, *P value <0.05. (d) Fibrosis quantification of GA muscle cross-sections 5 days after injury. The fibrotic index represents the percentage of the injury area occupied by connective tissue. Data represent mean±s.e.m. (n=4 WT and n=4 KO). Two-tailed unpaired Student’s t-test, *P value <0.05. (e) Representative micrograph of perilipin immunostaining on 5-day-cardiotoxin-injured GA muscle cross-section. Scale bar represents 50 μm. (f,g) Adipocyte numbers (perilipin-positive cells) per injured (f) or un-injured (g) area from WT and Ot KO mice. Data represent mean±s.e.m. (n=3 WT and n=5 KO). Two-tailed unpaired Student’s t-test, NS: not significant. Full size image

Moreover, similarly to 24-month-old C57/BL6 old mice, 12-month-old Ot KO mice display prematurely increased fibrosis when compared with age-matched WT littermates (Fig. 6d, Supplementary Fig. 1b). Appearance of adipocytes within the injured area has been reported to occur after muscle injury36,37,38,39. The number of adipocytes within the recently regenerated or un-injured muscle (assayed by perilipin staining) was generally low and a nonsignificant increase was observed in Ot KO mice, as compared with WT animals (Fig. 6e–g). However, we observed a clear increase in perimuscular and intermuscular adipose tissue deposition around the hind limb muscles in Ot KO versus WT littermates (Fig. 7a), consistent with the overall adipose tissue mass increase17.

Figure 7: Mice lacking oxytocin develop premature sarcopenia. (a) Representative photographs of Ot KO and WT mice hind limb skeletal muscles (right leg) after the skin was carefully removed (WT and KO mice are presented side by side on each photograph). Visualization of perimuscular adipose tissue deposition (arrows) and of exposed intermuscular adipose tissue (arrowheads). (i) Ventral view, arrows indicate the adipose tissue on the internal part of the quadriceps. The star shows that Ot KO mice display increased posterior subcutaneous adipose tissue. (ii) Ventral view showing adipose tissue deposition over the quadriceps (top arrows) and the tibialis anterior (TA) (bottom arrows). (iii) Lateral view showing adipose tissue deposition covering the quadriceps (top arrows) and the TA and gastrocnemius (GA) (bottom arrows). (iv) Dorsal view showing the intermuscular adipose tissue deposition of the hind leg (arrowheads) and the adipose tissue covering the GA muscle (arrows). As compared with the WT littermates, the KO mice have an increase in fat tissue in all the studied muscle groups, and visibly reduced muscle, which is studied in more detail and quantified below. (b) TA and (c) GA muscles from 12-month-old WT or Ot KO mice were weighed. Data represent mean±s.e.m. (top), representative pictures (bottom). Two-tailed unpaired Student’s t-test, *P value <0.05, ***P value <0.001, n=5 WT and n=10 KO mice. (d) The surface area and (e) the minimum Feret’s diameter were measured using WT and Ot KO muscle cross-section stained with H&E. Data represent mean±s.e.m. (n=4 mice per group), two-way ANOVA with post hoc Bonferroni test, *P value <0.05, **P value <0.01, NS: not significant. Full size image

Muscle ageing is characterized by a deficiency in muscle regeneration after injury but, most importantly, by muscle atrophy and altered muscle function observed in older individuals and defined as sarcopenia1,2. Having demonstrated that a lack of OT resulted in the diminished regeneration of old muscle after injury we next sought to examine whether OT was involved in age-associated sarcopenia. To test this hypothesis, we assessed the mass of the gastrocnemius (GA) and the TA muscles of 12-month-old Ot KO mice and their WT littermates. As shown in Fig. 7b,c, Ot deficiency resulted in significantly smaller muscles: Ot KO mice displayed a 32% decrease in muscle mass for the TA and a 22% decrease for GA as compared with their WT littermates. We further compared the muscle histology of Ot KO versus WT mice by measuring muscle fibre surface area as well as the minimum Feret diameter of muscle cross-section. Remarkably, there was a significant decrease in both the surface area and the minimum Feret diameter in Ot KO as compared with WT mice (Fig. 7d,e). Importantly, no difference was observed in the fibre surface area or in the minimum Feret diameter between WT and Ot KO mice when compared at 3 months of age, demonstrating that the muscle atrophy observed in Ot-deficient mice at 12 months of age was not a consequence of a developmental defect (Fig. 7d,e). These data demonstrate that the KO in Ot is the first known genetic defect that results in premature sarcopenia and suggest that in the absence of OT, the non-significant but noticeable decline in muscle regeneration observed at 3 months of age might contribute to the decline in muscle fibre size and increased fibrosis and fat deposition by 12 months of age (Fig. 6b).