Date Published: March 26, 2018
Publisher: Public Library of Science
Author(s): Ean G. Phillips, Luke A. Beggs, Fan Ye, Christine F. Conover, Darren T. Beck, Dana M. Otzel, Payal Ghosh, Anna C. F. Bassit, Stephen E. Borst, Joshua F. Yarrow, William D. Phillips.
Sclerostin is a circulating osteocyte-derived glycoprotein that negatively regulates Wnt-signaling after binding the LRP5/LRP6 co-receptors. Pharmacologic sclerostin inhibition produces bone anabolic effects after spinal cord injury (SCI), however, the effects of sclerostin-antibody (Scl-Ab) on muscle morphology remain unknown. In comparison, androgen administration produces bone antiresorptive effects after SCI and some, but not all, studies have reported that testosterone treatment ameliorates skeletal muscle atrophy in this context. Our purposes were to determine whether Scl-Ab prevents hindlimb muscle loss after SCI and compare the effects of Scl-Ab to testosterone enanthate (TE), an agent with known myotrophic effects. Male Sprague-Dawley rats aged 5 months received: (A) SHAM surgery (T8 laminectomy), (B) moderate-severe contusion SCI, (C) SCI+TE (7.0 mg/wk, im), or (D) SCI+Scl-Ab (25 mg/kg, twice weekly, sc). Twenty-one days post-injury, SCI animals exhibited a 31% lower soleus mass in comparison to SHAM, accompanied by >50% lower soleus muscle fiber cross-sectional area (fCSA) (p<0.01 for all fiber types). Scl-Ab did not prevent soleus atrophy, consistent with the relatively low circulating sclerostin concentrations and with the 91–99% lower LRP5/LRP6 gene expressions in soleus versus tibia (p<0.001), a tissue with known anabolic responsiveness to Scl-Ab. In comparison, TE partially prevented soleus atrophy and increased levator ani/bulbocavernosus (LABC) mass by 30–40% (p<0.001 vs all groups). The differing myotrophic responsiveness coincided with a 3-fold higher androgen receptor gene expression in LABC versus soleus (p<0.01). This study provides the first direct evidence that Scl-Ab does not prevent soleus muscle atrophy in rodents after SCI and suggests that variable myotrophic responses in rodent muscles after androgen administration are influenced by androgen receptor expression.
The musculoskeletal decline resulting from spinal cord injury (SCI) is precipitated by the neurologic insult and reduced loading in the paralyzed limbs . However, the molecular signals that regulate muscle and bone loss after SCI require further elucidation. In our companion paper, we reported that stimulation of either the Wnt/β-catenin signaling pathway, via a monoclonal anti-sclerostin antibody (Scl-Ab), or the androgen signaling pathway, via testosterone-enanthate (TE), resulted in significant cancellous bone preservation in a rodent moderate-severe contusion SCI model, albeit via differing bone anabolic and antiresorptive mechanisms, respectively . These results suggest that Wnt/β-catenin signaling and androgen signaling represent potential pathways influencing SCI-induced bone loss. Herein, we report the effects of these agents on sublesional skeletal muscles that were obtained from the animals examined in our companion paper because the Wnt/β-catenin signaling pathway  and the androgen signaling pathway produce anabolic effects in muscle , at least in the non-neurologically-impaired state, and because there is increasing recognition of biochemical bone-to-muscle crosstalk, as a mechanism through which musculoskeletal tissue is co-regulated [5,6]. In addition, the evaluation of off-target tissue responses remains important in the context of determining the systemic safety and/or efficacy of preclinical pharmacologic agents.
Pharmacologic strategies targeting preservation of musculoskeletal integrity have the potential to improve physical rehabilitation efforts after SCI. The principal findings of this investigation were that Scl-Ab did not prevent soleus atrophy after SCI and did not alter mass of the sublesional non-weight-bearing LABC muscle complex. We have previously reported that Scl-Ab and TE both prevented SCI-induced cancellous bone loss in rodents, albeit via differing cellular mechanisms . Specifically, Scl-Ab increased osteoblast surface and cancellous bone formation, indicating direct bone anabolic effects, whereas TE reduced osteoclast surface with minimal effect on bone formation, indicating antiresorptive actions . Herein, we examined the effects of Scl-Ab and TE on muscle preservation in the same animals from our previous report. The inability of Scl-Ab to induce myotrophic actions likely occurred because (1) the muscles we examined exhibited >90% lower mRNA expression of the sclerostin co-receptors (LRP5/LRP6) in comparison to the tibia, a tissue in which Scl-Ab produced potent bone anabolic actions after SCI [2,10] and/or (2) sclerostin was not present in a sufficient concentration within the circulation to attenuate LRP5/6-mediated Wnt-signaling in vivo. In this regard, the Wnt/β-catenin signaling pathway is anabolic in skeletal muscle  and sclerostin co-incubation has been shown to abolish Wnt3a-mediated C2C12 differentiation in culture, demonstrating that sclerostin antagonizes Wnt-signaling in an isolated murine muscle cell line . However, LRP6 expression is fiber-type specific, with normal and atrophic human skeletal muscle expressing LRP6 only in type II fibers and C2C12 cells expressing LRP6 only in the presence of fast myosin , explaining the relatively low LRP6 mRNA expression that we observed in the soleus, a muscle comprised predominantly of type I fibers. In comparison, Tran et al reported >50% of cultured myoblasts from murine muscle were LRP5 positive, although, satellite cells for this experiment were isolated from the tibialis anterior , which is composed of predominantly type II fibers. Additionally, it is important to note that the sclerostin dose used by Huang et al  to abolish Wnt3a-signaling (100 ng/ml) was 200–300 times greater than the circulating concentrations present in our rodents, suggesting that supraphysiologic sclerostin concentrations may be necessary to inhibit LRP5/6-mediated Wnt-signaling in skeletal muscle, although, direct dose-response studies are needed to verify this contention. Interestingly, Battaglino et al reported that circulating sclerostin was elevated in individuals acutely after SCI, when bone loss is most severe , and Qin et al reported that mice with sclerostin gene deletion do not exhibit bone loss after SCI , indicating that elevated sclerostin influences bone loss after SCI. Others have also reported that Scl-Ab treatment completely prevented sublesional bone loss in the relative absence of mechanical loading [10,33,34,35], demonstrating the ability of Scl-Ab to protect against disuse-induced bone loss. Our findings complement these reports by providing the first evaluation of muscle morphology after Scl-Ab treatment, which supplies another layer of support indicating that the bone anabolic actions of Scl-Ab occur relatively independently of muscle strain, given that that no discernable differences in muscle fCSA or voluntary hindlimb locomotor recovery (BBB scores) were present among SCI and SCI+Scl-Ab groups. In contrast, the presence of musculoskeletal strain may be necessary for Scl-Ab to potentiate myotrophic responses, as has been reported with several other pharmacologic agents that produce anabolic responses only in loaded muscle [36,37]. Preliminary support for this contention is derived from our findings that indicate highly positive associations exist among BBB score and soleus type IIA fCSA (r = 0.914) and IIA/IIX fCSA (r = 0.931) in SCI+Scl-Ab animals, suggesting that the presence of mechanical strain influences the ability of sclerostin inhibition to promote myotrophic responses in type II fibers. As such, future research evaluating the anabolic potential of Scl-Ab in muscles with relatively high type II MHC expression remains warranted, especially when administered in combination with supplemental loading.
In summary, Scl-Ab produced no discernable benefit or detriment to soleus muscle morphology or fiber type distribution after moderate-severe contusion SCI, likely because (1) the sclerostin LRP5/LRP6 co-receptors are only minimally expressed in the rat soleus, which is composed predominantly of type I fibers, (2) circulating sclerostin may not have been present in sufficient concentration to attenuate LRP5/6-mediated Wnt-signaling in vivo, and/or (3) mechanical stimuli may be necessary for Scl-Ab to promote anabolic response in skeletal muscle, as has been reported with several other myotrophic agents [36,37]. In contrast, TE completely preserved mass of the non-weight-bearing LABC muscle complex, but only partially prevented soleus atrophy, with the differences among muscles likely being explained by the relatively higher AR expression in LABC. These results highlight the importance of selecting appropriate muscles and examining hormone receptor expression in target tissues of preclinical model species when initiating pharmacologic therapy intended to promote myotrophic effects.