In this study, we developed a highly specific and accurate multiplexed LC-MS/MS assay for measuring circulating concentrations of mature and propeptide forms of the muscle-derived protein, myostatin, and two of its inhibitors, FLRG and GASP-1, in human serum. Using this novel approach and a well-characterized population-based sample, we show that absolute and relative concentrations of myostatin and propeptide are higher in younger men than younger women, increase with age in women, but in fact decrease with age in men. Intriguingly, these age-associated changes result in much higher circulating myostatin and propeptide concentrations per unit of lean mass in older women than older men. We also demonstrate that circulating concentrations of FLRG and, to a lesser extent, GASP-1 increase similarly in women and men with age, particularly in the context of sarcopenia. Finally, we report that circulating concentrations of myostatin exhibit positive, but not robust, age-adjusted correlations with relative ASM in both sexes.

As highlighted here and previously [31, 32], there are several challenges to the specific and accurate measurement of circulating myostatin concentrations using traditional antibody-based approaches, such as RIA, ELISA, and Western blotting. Through chromatographic separation and the use of peptide sequences, or “fingerprints,” that are unique to and, in particular, distinct from GDF-11, LC-MS/MS provides a highly specific method to quantify myostatin. Using this approach, we observed myostatin levels of 8.6 ± 3.7 ng/ml in men and 6.7 ± 3.3 ng/ml in women. These concentrations are considerably lower than the mean values (26.7 to >100 ng/ml) reported in several recent studies of healthy adults using commercial ELISA kits [16, 18, 19, 33–35] but similar to the results obtained in healthy adults using an ELISA comprised of proprietary and presumably more specific, reagents ([15, 17]. In addition to improved specificity, LC-MS/MS has better sensitivity for quantifying low abundance proteins than antibody-based approaches. By coupling immunopurification and LC-MS/MS, we observed both a LOD and a LOQ of 0.01 nM, or 0.248 ng, for myostatin. In comparison, Peiris et al. observed that the LOD for recombinant myostatin proteins by Western blot was ~83.33 nM, or 2000 ng [32]. We established similarly low LOD and LOQ values for propeptide, FLRG, and GASP-1. Therefore, this novel multiplexed LC-MS/MS assay offers a highly specific and sensitive means to quantify circulating concentrations of myostatin and myostatin-related proteins in a single small (400 ul) sample of human serum.

Myostatin is a promising therapeutic target to improve muscle health [36]. Several pharmacological approaches have been developed, including neutralizing antibodies, propeptides, soluble decoy receptors, and receptor antagonists. Such interventions have been shown to increase skeletal muscle mass and improve parameters of strength and physical function in preclinical models of aging and disease [12, 37, 38]. A number of early phase clinical trials are underway [36, 39]. It is therefore surprising then, that relatively little is known about the relationship between circulating myostatin concentrations and skeletal muscle mass in human conditions associated with its loss or degeneration. Indeed such data could inform the selection of indications or individuals that may be most responsive to targeted interventions. In the present study, we demonstrate contrasting age-associated changes in myostatin and propeptide levels in women and men. Specifically, we observed higher absolute and relative circulating concentrations in older women compared to younger women and lower concentrations in older men compared to younger men. Unexpectedly, we also measured higher concentrations of myostatin and propeptide per unit of lean mass in older women and sarcopenic older women than in corresponding groups of men. The prevalence of sarcopenia is higher in women than men; however, we can only speculate that myostatin plays a causal role in age-associated muscle loss in women and that women may be more responsive than men to anti-myostatin therapies. In both sexes, we failed to see meaningful differences in myostatin concentrations or the ratio of myostatin to propeptide between older subjects and sarcopenic older subjects. Of note, we studied healthy older persons without chronic diseases associated with the deterioration of skeletal muscle, including cancer, chronic heart failure, chronic obstructive pulmonary disease, diabetes, chronic kidney disease, and human immunodeficiency virus. Future research is needed to determine the extent to which myostatin concentrations, or the ratio of myostatin to propeptide, are associated with skeletal muscle mass and function in the context of such conditions.

In 1962, Bullough and Lawrence first proposed that “diffusible substances,” or chalones, regulate the mass of the specific tissue from which they were derived [1, 40]. Shortly after its discovery as a protein synthesized and secreted by skeletal muscle, Lee and McPherron highlighted the potential for myostatin to be a muscle chalone [41]. As a chalone, myostatin may be an evolutionarily conserved mechanism that was selected to prevent the allocation of limited resources to the further development and maintenance of the tissue from which it is derived. In younger women, this may have been critical for reproduction. Consistent with the concept of antagonistic pleiotropy, this early life benefit of myostatin may have later life costs, namely, the excessive deterioration of skeletal muscle. With the significant extension in life expectancy beyond the menopause, it is plausible that counter regulatory mechanisms could not be selected for and, consequently, myostatin contributes to the age-associated loss of skeletal muscle in women. In men on the other hand, myostatin is highest in younger men with the greatest muscle mass and, as would be anticipated for a chalone, lowest in older men with the least muscle mass. This same pattern is observed for sclerostin, which is synthesized in and secreted by osteocytes. Sclerostin functions as a potent negative regulator of the Wnt/β-catenin signaling pathway to inhibit bone formation [42]. Serum sclerostin levels are positively associated with total body bone mass in both women and men [43]. The reason for sexually dimorphic age-related changes in myostatin is unclear. We observed that FLRG and GASP-1 are higher in younger women than younger men and increase with age in both sexes and even more so in those who are sarcopenic. This is the first report of FLRG and GASP-1 serum concentrations in women; however, in a smaller study using ELISAs, Ratvekius et al. observed no differences or trends for a decrease in FLRG and GASP-1 in older men compared to younger men. Furthermore, our data fail to show meaningful relationships between circulating myostatin concentrations in women and established mediators of skeletal muscle mass, including bioavailable testosterone, IGF-1, and IGF-2. In men, we observed positive, not negative, associations between circulating myostatin and bioavailable estrogen and testosterone concentrations. Additional research is needed to further understand what factors regulate myostatin abundance and/or activity and how such factors are affected by aging in both women and men.

Our study provides important insights into age-related changes and sex differences in the circulating concentrations of myostatin and its related proteins in healthy adults. However, it is important to recognize the cross-sectional design of our study. Longitudinal studies are needed to better define the relationships between myostatin, propeptide, FLRG and GASP-1 and age- and disease-related changes in skeletal muscle mass and performance, and the utility of these proteins as a biomarkers of current and future muscle health. Our sample was also predominantly white, and underrepresented with respect to persons of African, Asian, Hispanic, Latino, American Indian, and Alaskan Native ancestry groups. To our knowledge, the influence of origin on myostatin concentrations has not been investigated. Furthermore, our assay has notable strengths, including the ability to precisely monitor four analytes in a single sample of merely 400 ul of serum. However, while we can specifically monitor the abundance of C-terminal (mature) and N-terminal (propeptide) regions unique to myostatin, at this time, we are not able to define the stoichiometry of free (active) versus bound (latent) forms in vivo. Of note, we did attempt an acid activation step in pooled serum to overcome this hurdle; however, we had reduced recovery of all proteins with the exception of propeptide, which did not change. We therefore chose to immunoprecipitate under physiological conditions without acid activation. Even so, we believe this multiplexed LC-MS/MS approach represents the current upper limit of specificity and sensitivity for assessing myostatin, propeptide, FLRG, and GASP-1 in human clinical samples, and that our study represents the most comprehensive assessment of these proteins in both women and men to date.