Myostatin is a member of the transforming growth factor (TGF-) superfamily and is a negative regulator of skeletal muscle growth. When myostatin is missing or non-functional, the result is uncontrolled muscle growth with a concomitant increase in bone strength. When mice lacking the myostatin gene (mstn/mstn) were subjected to running on a treadmill, their bone strength increased above non-exercised mstn/mstn mice. Exercise has also been shown to be beneficial for human patients at an increased risk for fracture (i.e. osteoporosis, osteogenesis imperfecta). Based on these previous reports, this project was designed to determine if a combination of myostatin inhibition and exercise could improve the bone strength in G610C mice with compromised bone.

Project Aims

1. Determine if a reduction and/or complete absence of the myostatin protein will ameliorate the bone phenotype of heterozygote and homozygote G610C mice; and

2. Determine if exercise coupled with reduction and/or absence of myostatin will increase muscle mass and bone strength in heterozygote and homozygote G610C mice.

Additionally, after the initiation of this project, it was reported that the myostatin genotype of the mother affects the muscle weight of the offspring. Wildtype pups born to mstn/+ mothers had increased muscle weight as compared to those born to wildtype mothers. The same was true for mstn/+ pups.

At four months of age, femurs and tibiae from male and female wildtype, heterozygote (mstn/+) and homozygote (mstn/mstn) myostatin knock-out were removed along with several muscles. Total body mass was found to be greater in mstn/mstn mice as compared to wildtype. Males were also larger than females in all genotypes. Muscle mass was larger in mstn/mstn mice than in wildtype in both males and females. Gender also impacted muscle mass with males having larger muscles masses than females. Geometry was measured via CT analysis which demonstrated gender differences in all parameters measured, including polar moment of area, with males having longer and wider bones. Additionally, femurs from male mstn/mstn mice had a larger polar moment of area then femurs from either wildtype or mstn/+ mice. Torsional loading to failure also demonstrated gender differences in whole bone strength and stiffness with males having stronger and stiffer bones than females. Additionally, mstn/mstn males were also significantly stronger and stiffer than male wildtype or mstn/+ mice. Bone material properties were not different between the genotypes in either male or female mice. Taken together, the data suggest that myostatin inhibition impacts femoral geometry and increases whole bone strength and stiffness without impacting bone material in a gender-specific manner.

Due to the complex nature of these analyses with the addition of gender differences and allele status of the mother, we have revised our original breeding scheme. We are currently breeding mstn/+ mice with G610C/+ mice to generate mice of four genotypes: wildtype, mstn/+ mice, G610C/+ mice and mstn/+ G610C/+ mice. Two types of breeding pairs have been set up: 1) mstn/+ mother and G610C/+ father and 2) G610C/+ mother and mstn/+ father. Offspring from these crosses will be divided into two groups, controls and exercisers. Hindlimb muscles (soleus, plantaris, tibialis anterialis and gastrocnemius) and bones from both groups will be analyzed. Femora and tibiae will be analyzed via CT analysis and torsional loading to failure to determine bone geometry and strength. Muscles will be analyzed for contractile generating capacity, gross pathology and cross-sectional area. Collagen content will also be measured using the hydroxyproline assay in the femora from both groups. Data will be separated based on both gender and maternal myostatin allele status.

Earth-based Applications of Research Project
Muscle atrophy and bone loss are both associated with the weightlessness experienced by astronauts, but these are also widespread health concerns on Earth. As a person ages, muscle use tends to decrease. With disuse, muscle atrophy increases and bone strength decreases with a concomitant increase in fracture risk. While it has long been known that exercise decreases muscle atrophy and fracture risk while increasing bone strength, the role that myostatin inhibition may play in further augmenting the positive physiological effects of exercise is not known. This study is designed to determine what impact, if any, myostatin inhibition has on bone strength as well as on muscle physiology and strength in both normal mice and mice with compromised bone whose clinical outcome mimics that seen in osteoporosis patients. Additionally, the effects of exercise on the bone strength in both the normal mice and the mice with compromised bone will be evaluated to determine if the combination of myostatin inhibition and exercise will prevent both muscle atrophy and bone loss. At the completion of this study, we will have determined whether a pharmacological approach of myostatin inhibition should be pursued, along with the inclusion of exercise, to potentially ameliorate the muscle atrophy and bone loss often seen as a consequence of a sedentary lifestyle.

Project Description
NASA Task Book Entry