Intensive efforts have been made over the last decade to explore the molecular mechanisms underlying the regulation and function of myostatin, a key negative regulator of skeletal muscle growth. The identification of several components of the myostatin-signalling pathway had important implications with respect for testing the therapeutic value of a myostatin antagonist in muscle wasting disorders such as Duchenne muscular dystrophy [22
] cachexia and age-related sarcopenia [24
]. Among the known myostatin-binding proteins, GASP1 has been shown to inhibit myostatin activity in vitro
and may maintain myostatin latency but no data was available on the effect of GASP1 when expressed as a transgene in all skeletal muscles of wild type mice. In the present study, we have generated a “gain of function” mouse model to further understand the in vivo
roles of Gasp1
. These mice carry a transgene containing the Gasp1
coding sequence under the transcriptional regulation of the ubiquitous CMV promoter. As a consequence, Gasp1
mRNA expression is greatly enhanced in several organs including muscle, brain, spleen, liver, heart, lung and kidney. Analysis of the two Gasp1
transgenic lines with the highest transgene expression revealed a skeletal muscle hypertrophic phenotype. This might have been expected based on previous data indicating that GASP1 can function as a myostatin antagonist, similar to follistatin or the follistatin-related gene protein whose overexpression leads to an increase of the muscle mass. Furthermore, viral delivery of a Gasp1
expression cassette into adult muscle has been shown to induce increases in muscle mass and grip strength [13
]. The surGasp1 mice have similar average life span compared to standard inbred laboratory mice (http://research.jax.org/faculty/harrison/ger1vi_Lifespan.html
) Except the phenotype described in this paper, no other gross abnormalities were noted, even in elder surGasp1 mice (≈ 28 months). Effects of elevated GASP1 on body growth were not observed before day 15 of postnatal life. The enhanced muscle growth occurs in both male and female animals with a more pronounced phenotype in male pectoralis major
muscle. The individual weights of the gastrocnemius
, rectus femoris
and pectoralis major
muscles were increased. The masses of other skeletal muscles were also increased. The sizes of other internal organs did not differ from those of control mice despite overexpression of GASP1. Unlike the myostatin-deficient animals or FS overexpressing mice, which exhibit both muscle hypertrophy and hyperplasia, we only observed an increase in myofiber size without a corresponding increase in myofiber number in surGasp1 animals. As the number of myofibers in muscle is largely determined during prenatal development, the overexpression of Gasp1
does not seem to provide prenatal effect. However, we cannot preclude that the lack of hyperplasia is not the result of a too moderate expression of the transgene during embryonic and fetal stages, although Gasp1
is strongly expressed in postnatal 3 days mice (Additional file 2
). In the litterature, heterozygous mutations in the myostatin gene or surexpression of its propeptide have been reported to result also in hypertrophy and no hyperplasia [27
]. Taken together, these results may be reflective of an incomplete inhibition of the myostatin. Moreover, Zhu et al.
] have described that mice carrying a dominant negative form of myostatin preventing the release of the mature myostatin from the propeptide exhibited a significant increase in muscle mass that resulted from myofiber hypertrophy and not from myofiber hyperplasia. As Hill et al
] suggested, GASP1 may inhibit the propeptide proteolysis to keep the myostatin in a latent and inactive form. Such possible mechanism may explain the observed phenotype in the surGasp1 mice.
The body weight increment in surGasp1 mice is visible at 3 weeks of age. However, during animal growth, the body weight difference between wild-type and transgenic mice does not increase, indicating that the effect of Gasp1
overexpression may arise during the first 21 days of life, probably by satellite cells activity or AKT/FOXO dependent signalling pathway . To gain insights into the molecular mechanism, we performed an expression array analysis of 91 genes involved in muscle development. From those, only 5 genes Ccnd1
(cyclin D1), Actvr1c
were slightly down- or up-regulated in the surGasp1 mice (Figure , Additional file 3
: Table 1). Cyclin D1, an important component of the cell cycle machinery has been shown to be a major intracellular target for myostatin during myostatin-induced G1 cell cycle arrest and proliferation inhibition [29
]. Ji et al.
] showed that myostatin blocked the recruitment of p300 to the cyclin D1 promoter, resulting in the silencing of cyclin D1 gene expression. The increase of Ccnd1
expression level in the surGasp1 mice is in accordance with those previous studies. The analysis of the Gasp1
promoter revealed the presence of four PPARγ binding sites. A negative feedback loop may exist and could explain the down-regulation of Ppar
γ in the mutant mice. Finally, most of the observed variations are related to an activation of the canonical Wnt/β-catenin signalling pathway and are consistent with observations in GDF8-null mice [31
]. The mRNA expression levels of Pax3
genes which are expressed by postnatal satellite cells [32
] or members of AKT/FOXO pathway remained unchanged in surGasp1 mice when compared to wildtype. However, the activation or repression of this pathway is essentially due to postranslational modification. Further studies are needed to understand the relevance of the AKT/FOXO signalling in the surGasp1 phenotype.
Figure 8 Relative expression levels of genes involved in muscle development in quadriceps surGasp1 mice. Using the TLDA technology, mRNA expression levels were determined by qRT–PCR relative to the reference genes Gapdh, TFIID, Dffa and fcgrt in quadriceps (more ...)
In mammals, myofibers are mainly classified into glycolytic and oxidative fibers based on their metabolic profiles. In mice, fast glycolytic fibers express the type IIB MHC isoform whereas oxidative fibers express type I (slow fibers), the fibers expressing MHC IIA are capable of both oxidative and glycolytic metabolism. We could show that Gasp1
overexpression does not significantly change fiber type distribution while a lack of myostatin results in an alteration in the fiber type composition [33
]. Glycolytic (phosphofructokinase, lactate dehydrogenase) or oxidative (citrate synthase, isocitrate dehydrogenase, cytochrome c-oxidase) enzyme activities did not show any significant differences between wild-type and surgasp1-20 mice, confirming the above mentioned result (data not shown).
Interestingly, we do not detect a change in fat pad mass in our Gasp1
transgenic mice. This result differs from the results reported for the myostatin knockout mice or transgenic mice overexpressing the myostatin propeptide in which a reduction in adiposity is observed [14
]. Recent literature showed that myostatin inhibition in skeletal muscle, but not in adipose tissues, is primarily responsible for a decrease of fat mass [39
]. This effect on fat pad could reflect a regulation of myostatin independant from GASP1.