Effects of maternal immunization against myostatin on skeletal muscle mass of offspring in mice

Abstract

Myostatin (growth & differentiation factor-8), a member of the transforming growth factor-β superfamily, is a potent negative regulator of skeletal muscle growth. Previous studies have demonstrated hypermuscularity being induced by the absence or suppression of functional myostatin and conversely, muscle atrophy upon myostatin overexpression or administration. These studies indicate that myostatin-blockade has potential applications in improving muscle mass in farm animals and in treating muscle atrophies in humans. Since immunoneutralization has been effective in inhibiting biological molecules, it was hypothesized that maternal immunization against myostatin in female mice might enhance muscle mass in offspring via myostatin-blockade in the developing embryos and neonates by maternally transferred anti-myostatin antibody. In this study, twenty reproducing, twelve week-old female mice were divided into four groups, and immunized against KLH (Control) or recombinant porcine mature myostatin (rMyo) or two different KLH-conjugated myostatin peptides (Myo-I and Myo-2). Animals were immunized subcutaneously with 0.2 mg of immunogen mixed in TiterMax® adjuvant. Two or three boosters were administered prior to mating to ensure that the immunized female mice had significant antibody titers against their respective immunogens in ELISA. Sera collected from 3-day old neonates demonstrated significant titers, confirming the maternal transfer of anti-myostatin antibodies to offspring. However, no significant effects of immunization on the body weights of offspring were observed during 8 weeks of growout period. The offspring of the Myo-2 group had significantly heavier gastrocnemius and triceps muscles at 8 weeks compared to the controls (P<0.01), but the muscle mass of the offspring from the rMyo and Myo-l groups did not significantly differ from that of the control offspring. Since it is possible that the antibodies generated against three different antigens may have different binding characteristics and ability to inhibit myostatin activity, we examined the binding affinities of the antibodies to myostatin using Western blot analysis and the ability of the antibodies to suppress myostatin activity using pGL3 (CAGA) 12-Luc-luciferase reporter system. The antibodies from the three treatment groups showed affinity to mature myostatin monomer, but none of the antibodies showed affinity to myostatin dimer. In the pGL3 (CAGA) 12-Luc-luciferase reporter assay system, none of the antibodies from the three immunized groups showed the ability to suppress the biological activity of myostatin. In conclusion, we observed that the active immunization against myostatin using Myo-2 peptide improved the muscle growth of offspring. However, the inability of the anti-Myo-2 antibody to bind mature myostatin dimer and to suppress the myostatin's activity led us to question whether the improved muscle mass in the Myo-2 offspring was due to the inhibition of myostatin's biological activity by the anti-Myo-2 antibodies. Therefore, further studies are needed to determine whether active immunization against myostatin has the potential to improve skeletal muscle growth of offspring.