Inhibiting TGF-ß activity improves respiratory function in mdx mice.

Respiratory function is the main cause of mortality in patients with Duchenne muscular dystrophy (DMD). Elevated levels of TGF-ß play a key role in the pathophysiology of DMD. To determine whether therapeutic attenuation of TGF-ß signaling improves respiratory function, mdx mice were treated from 2 weeks of age to 2 months or 9 months of age with either 1D11 (a neutralizing antibody to all three isoforms of TGF-ß), losartan (an angiotensin receptor antagonist), or a combination of the two agents. Respiratory function was measured in nonanesthetized mice by plethysmography. The 9-month-old mdx mice had elevated Penh values and decreased breathing frequency, due primarily to decreased inspiratory flow rate. All treatments normalized Penh values and increased peak inspiratory flow, leading to decreased inspiration times and breathing frequency. Additionally, forelimb grip strength was improved after 1D11 treatment at both 2 and 9 months of age, whereas, losartan improved grip strength only at 2 months. Decreased serum creatine kinase levels (significant improvement for all groups), increased diaphragm muscle fiber density, and decreased hydroxyproline levels (significant improvement for 1D11 only) also suggested improved muscle function after treatment. For all endpoints, 1D11 was equivalent or superior to losartan; coadministration of the two agents was not superior to 1D11 alone. In conclusion, TGF-ß antagonism may be a useful therapeutic approach for treating DMD patients.

Diversity of stonefly hexamerins and implication for the evolution of insect storage proteins.

Hexamerins are large storage proteins of insects in the 500 kDa range that evolved from the copper-containing hemocyanins. Hexamerins have been found at high concentration in the hemolymph of many insect taxa, but have remained unstudied in relatively basal taxa. To obtain more detailed insight about early hexamerin evolution, we have studied hexamerins in stoneflies (Plecoptera). Stoneflies are also the only insects for which a functional hemocyanin is known to co-occur with hexamerins in the hemolymph. Here, we identified hexamerins in five plecopteran species and obtained partial cDNA sequences from Perla marginata (Perlidae), Nemoura sp. (Nemouridae), Taeniopteryx burksi (Taeniopterygidae), Allocapnia vivipara (Capniidae), and Diamphipnopsis samali (Diamphipnoidae). At least four distinct hexamerins are present in P. marginata. The full-length cDNA of one hexamerin subunit was obtained (PmaHex1) that measures 2475 bp and translates into a native polypeptide of 702 amino acids. Phylogenetic analyses showed that the plecopteran hexamerins are monophyletic and positioned at the base of the insect hexamerin tree, probably diverging about 360 million years ago. Within the Plecoptera, distinct hexamerin types evolved before the divergence of the families. Mapping amino acid compositions onto the phylogenetic tree shows that the accumulation of aromatic amino acids (and thus the evolution of "arylphorins") commenced soon after the hexamerins diverged from hemocyanins, but also indicates that hexamerins with distinct amino acid compositions reflect secondary losses of aromatic amino acids.

Regulation of muscle growth by multiple ligands signaling through activin type II receptors.

Myostatin is a secreted protein that normally functions as a negative regulator of muscle growth. Agents capable of blocking the myostatin signaling pathway could have important applications for treating human muscle degenerative diseases as well as for enhancing livestock production. Here we describe a potent myostatin inhibitor, a soluble form of the activin type IIB receptor (ACVR2B), which can cause dramatic increases in muscle mass (up to 60% in 2 weeks) when injected into wild-type mice. Furthermore, we show that the effect of the soluble receptor is attenuated but not eliminated in Mstn(-/-) mice, suggesting that at least one other ligand in addition to myostatin normally functions to limit muscle growth. Finally, we provide genetic evidence that these ligands signal through both activin type II receptors, ACVR2 and ACVR2B, to regulate muscle growth in vivo.

Loss of myostatin expression alters fiber-type distribution and expression of myosin heavy chain isoforms in slow- and fast-type skeletal muscle.

Myostatin (Mstn) is a member of the transforming growth factor-beta family that negatively regulates skeletal muscle mass. Mstn knockout mice have greater skeletal muscle mass than wild-type littermates. We investigated the effect of Mstn on fiber type by comparing adult muscles from the murine Mstn knockout with wild-type controls. Based on myofibrillar ATPase staining, the soleus of Mstn knockout mice displays a larger proportion of fast type II fibers and a reduced proportion of slow type I fibers compared with wild-type animals. Based on staining for succinate dehydrogenase (SDH) activity, a larger proportion of glycolytic fibers and a reduced proportion of oxidative fibers occur in the extensor digitorum longus (EDL) of Mstn knockouts. These differences in distribution of fiber types are accompanied by differences in the expression of myosin heavy chain (MHC) isoforms. In both Mstn knockout soleus and EDL, larger numbers of faster MHC isoforms are expressed at the expense of slower isoforms when compared with wild-type littermates. Thus, the absence of Mstn in the knockout mouse leads to an overall faster and more glycolytic muscle phenotype. This muscle phenotype is likely a consequence of developmental processes, and inhibition of Mstn in adults does not cause a transformation to a more fast and glycolytic phenotype. Our findings suggest that myostatin has a critical role in regulating the formation, proliferation, or differentiation of fetal myoblasts and postnatal fibers.

Inhibition of myostatin in adult mice increases skeletal muscle mass and strength.

A human therapeutic that specifically modulates skeletal muscle growth would potentially provide a benefit for a variety of conditions including sarcopenia, cachexia, and muscular dystrophy. Myostatin, a member of the TGF-beta family of growth factors, is a known negative regulator of muscle mass, as mice lacking the myostatin gene have increased muscle mass. Thus, an inhibitor of myostatin may be useful therapeutically as an anabolic agent for muscle. However, since myostatin is expressed in both developing and adult muscles, it is not clear whether it regulates muscle mass during development or in adults. In order to test the hypothesis that myostatin regulates muscle mass in adults, we generated an inhibitory antibody to myostatin and administered it to adult mice. Here we show that mice treated pharmacologically with an antibody to myostatin have increased skeletal muscle mass and increased grip strength. These data show for the first time that myostatin acts postnatally as a negative regulator of skeletal muscle growth and suggest that myostatin inhibitors could provide a therapeutic benefit in diseases for which muscle mass is limiting.