Myostatin (GDF-8) is the chief chalone in skeletal muscle and negatively controls adult skeletal muscle growth. The role of myostatin during overload-induced hypertrophy of adult muscle is unclear. We tested the hypothesis that overloaded adult rodent skeletal muscle would result in reduced myostatin protein levels. Overload-induced hypertrophy was accomplished by unilateral tenotomy of the gastrocnemius tendon in male adult Sprague-Dawley rats followed by a two-week period of compensatory overload of the plantaris and soleus muscles. Western blot analysis was performed to evaluate changes in active, latent and precursor myostatin protein levels. Significant hypertrophy was noted in the plantaris (494 ± 29 vs. 405 ± 15 mg, p < 0.05) and soleus (289 ± 12 vs. 179 ± 37 mg, p < 0.05) muscles following overload. Overloaded soleus muscle decreased the concentration of active myostatin protein by 32.7 ± 9.4% (p < 0.01) while the myostatin precursor protein was unchanged. Overloaded plantaris muscle decreased the concentration of active myostatin protein by 28.5 ± 8.5% (p < 0.01) while myostatin precursor levels were reduced by 17.5 ± 5.9% (p < 0.05). Myostatin latent complex concentration decreased in the overloaded soleus and plantaris muscle by 15.0 ± 5.9% and 70.0 ± 2.3% (p < 0.05), respectively. These data support the hypothesis that the myostatin signaling pathway in overloaded muscles is generally downregulated and contributes to muscle hypertrophy. Plasma concentrations of total and active myostatin proteins were similar in overloaded and control animals and averaged 8865 ± 526 pg/ml and 569 ± 28 pg/ml, respectively. Tissue levels of BMP-1, an extracellular proteinase that converts myostatin to its active form, also decreased in overloaded soleus and plantaris muscles by 40.4 ± 12.9% and 32.9 ± 6.9% (p < 0.01), respectively. These data support the hypothesis that local, rather than systemic, regulation of myostatin contributes to the growth of individual muscles, and that an association exists between the extracellular matrix proteinase BMP-1 and the amount of active myostatin in overloaded muscles.

Les protéases Tolloïdes constituent un groupe de métalloprotéases extracellulaires comptant quatre membres chez les mammifères (BMP-1, mTLD, mTLL-1 et mTLL-2). Ces protéases jouent un rôle majeur dans le développement et la réparation tissulaire, ainsi que dans certaines pathologies comme les fibroses. En 2006, le premier inhibiteur endogène des protéases Tolloïdes a été identifié chez le xénope et le poisson zèbre. Il s'agit de la protéine Sizzled, qui appartient à la famille des secreted Frizzled-Related proteins (sFRPs). Le travail présenté dans ce manuscrit suggère que ce mécanisme d'inhibition des protéases Tolloïdes par les sFRPs n'est pas conservé chez les mammifères. En effet, trois des cinq sFRPs de mammifères ont été testées (sFRP1, sFRP2 et sFRP4), et aucune d'entre elles ne s'est avérée capable d'inhiber l'activité de la protéase BMP-1 humaine in vitro. Ce travail montre toutefois que les protéases BMP-1, mTLD et mTLL-1 humaines peuvent être inhibées de façon puissante et spécifique par la protéine Sizzled de xénope. Cette inhibition repose sur l'interaction du domaine Frizzled de Sizzled avec le domaine catalytique des protéases Tolloïdes. Plus particulièrement, les résidus Asp-92, Phe-94, Ser-43 et Glu-44 de Sizzled (dont certains ne sont pas présents chez les sFRPs de mammifères) jouent un rôle crucial dans cette inhibition. Enfin, nous nous sommes intéressés au variant long du collagène XVIII, qui comporte également un domaine Frizzled. Nous avons pu montrer que BMP-1 clive le collagène XVIII in vitro, libérant un fragment contenant le domaine Frizzled. Des expériences sont en cours pour déterminer si ce fragment est capable d'inhiber les protéases Tolloïdes / Tolloid proteinases constitute a group of extracellular metalloproteinases which includes four members in mammals (BMP-1, mTLD, mTLL-1, mTLL-2). These proteinases play major roles in development, tissue repair and related pathological conditions such as fibrosis. In 2006, the first endogenous inhibitor of Tolloid proteinases was identified in Xenopus and zebrafish. This inhibitor, called Sizzled, is a member of the secreted Frizzled- related proteins (sFRPs). The present study strongly suggests that inhibition of Tolloid proteinases activity by sFRPs is not conserved in mammals. Indeed, three of the five mammalian sFRPs were tested (sFRP1, sFRP2 and sFRP4) and none of them was found to inhibit human BMP-1 activity in vitro. In contrast, this study demonstrates that Xenopus Sizzled is a potent and specific inhibitor of human BMP-1, mTLD and mTLL-1. This inhibition involves an interaction between the Frizzled domain of Sizzled and the catalytic domain of Tolloid proteinases. More precisely, residues Asp-92, Phe-94, Ser-43 and Glu-44 of Sizzled (among which only Asp-92 is conserved in mammalian sFRPs) play a crucial role in Tolloid proteinase inhibition. Finally, we studied the longest isoform of collagen XVIII, which also contains a Frizzled domain. We found that BMP-1 can cleave collagen XVIII in vitro, resulting in a Frizzled domain-Containing fragment. Experiments are in progress to determine if this fragment can also inhibit Tolloid proteinase activity