Highly Specific Detection of Myostatin Prodomain by an Immunoradiometric Sandwich Assay in Serum of Healthy Individuals and Patients

<div><p>Background</p><p>Myostatin is a muscle derived factor that functions as a negative regulator of skeletal muscle growth. Induction of myostatin expression was observed in rodent models of muscle wasting and in cachectic patients with cancer or pulmonary disease. Therefore, there is an increasing interest to use serum myostatin as a biomarker.</p> <p>Methods</p><p>We established an immunoradiometric sandwich assay (IRMA), which uses a commercially available chicken polyclonal, affinity purified antibody directed against human myostatin prodomain. We determined the serum concentrations of myostatin prodomain in 249 healthy individuals as well as 169 patients with heart failure, 53 patients with cancer and 44 patients with chronic pulmonary disease.</p> <p>Results</p><p>The IRMA had a detection limit of 0.7ng/ml, an intraassay imprecision of ≤14.1% and an interassay imprecision of ≤ 18.9%. The specificity of our assay was demonstrated by size exclusion chromatography, detection of myostatin by Western-blotting and a SMAD-dependent transcriptional-reporter assay in the signal-rich serum fractions, as well as lack of interference by unspecific substances like albumin, hemoglobin or lipids. Myostatin prodomain was stable at room temperature and resistant to freeze-thaw cycles. Apparently healthy individuals over the age of 55 had a median myostatin prodomain serum concentration of 3.9ng/ml (25^th-75^th percentiles, 2-7ng/ml) and we could not detect increased levels in patients with stable chronic heart failure or cancer related weight loss. In contrast, we found strongly elevated concentrations of myostatin prodomain (median 26.9ng/ml, 25^th-75^th percentiles, 7-100ng/ml) in the serum of underweight patients with chronic pulmonary disease.</p> <p>Conclusions</p><p>We established a highly specific IRMA for the quantification of myostatin prodomain concentration in human serum. Our assay could be useful to study myostatin as a biomarker for example in patients with chronic pulmonary disease, as we detected highly elevated myostatin prodomain serum levels in underweight individuals of this group.</p> </div

Myostatin prodomain serum levels in patients compared to healthy individuals.

<p>Myostatin prodomain serum concentrations of apparently healthy individuals <55 (n=63) or ≥55 years of age (n=186), in patients suffering from heart failure (CHF, n=169), gastrointestinal or hepatic cancer (GI-Ca, n=53) and chronic pulmonary disease (CPD, n=44). Data are presented as box (25^th percentile, median, and 75^th percentile) and whisker (10^th and 90^th percentiles) plots.</p

Mean calibration curve of the myostatin prodomain immunoradiometric sandwich assay.

<p>The mean calibration curve of 20 independent assay runs is shown. The x-axis is displayed as logarithmic scale. The values on the y-axis are presented as mean with the 95% confidence limits.</p

Myostatin prodomain serum levels in healthy individuals.

<p><b>A</b> Myostatin prodomain serum concentrations in apparently healthy individuals ≤25 (n=33), 26-55 (n=46), 56-65 (n=138) and ≥66 (n=32) years of age. <b>B</b> Myostatin prodomain serum concentration in apparently healthy male and female individuals ≥55 years of age. Data are presented as box (25^th percentile, median, and 75^th percentile) and whisker (10^th and 90^th percentiles) plots.</p

Specificity of the myostatin prodomain immunoradiometric sandwich assay.

<p><b>A</b> Size-exclusion chromatography of two serum samples analyzed for myostatin prodomain. The dotted line represents the serum with high myostatin prodomain concentration (1629ng/ml), while the continuous line represents the serum with the low myostatin prodomain concentration (1.37ng/ml). B SMAD (CAGA) luciferase reporter assay showing acid induced myostatin activity in different serum fractions of one serum with high and one with low myostatin concentration as determined by our IRMA C Western-blot showing myostatin prodomain levels (arrow, 25kDa) as detected by the propeptide (=prodomain) specific antibody MAB-7881 (R&D systems). Low/high myostatin denotes serum with low and high myostatin prodomain concentrations. D Western-blot showing myostatin ligand dimers (arrow, 25kDa) as detected by the myostatin ligand specific antibody AB3239 (Millipore). + denotes positive control (recombinant myostatin ligand, R&D systems), the arrow head marks the localization of monomeric myostatin ligand (12.5 kDa). Low/high myostatin denotes serum with low and high myostatin prodomain concentrations.</p