Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides

Chimeric RNA/DNA oligonucleotides (“chimeraplasts”) have been shown to induce single base alterations in genomic DNA both in vitro and in vivo. The mdx mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated “MDX1”) designed to correct the point mutation in the dystrophin gene in mdx mice. After direct injection of MDX1 into muscles of mdx mice, immunohistochemical analysis revealed dystrophin-positive fibers clustered around the injection site. Two weeks after single injections into tibialis anterior muscles, the maximum number of dystrophin-positive fibers (approximately 30) in any muscle represented 1–2% of the total number of fibers in that muscle. Ten weeks after single injections, the range of the number of dystrophin-positive fibers was similar to that seen after 2 wk, suggesting that the expression was stable, as would be predicted for a gene-conversion event. Staining with exon-specific antibodies showed that none of these were “revertant fibers.” Furthermore, dystrophin from MDX1-injected muscles was full length by immunoblot analysis. No dystrophin was detectable by immunohistochemical or immunoblot analysis after control chimeraplast injections. Finally, reverse transcription–PCR analysis demonstrated the presence of transcripts with the wild-type dystrophin sequence only in mdx muscles injected with MDX1 chimeraplasts. These results provide the foundation for further studies of chimeraplast-mediated gene therapy as a therapeutic approach to muscular dystrophies and other genetic disorders of muscle.

Increased glycogen accumulation in transgenic mice overexpressing glycogen synthase in skeletal muscle.

To investigate the role of glycogen synthase in controlling glycogen accumulation, we generated three lines of transgenic mice in which the enzyme was overexpressed in skeletal muscle by using promoter-enhancer elements derived from the mouse muscle creatine kinase gene. In all three lines, expression was highest in muscles composed primarily of fast-twitch fibers, such as the gastrocnemius and anterior tibialis. In these muscles, glycogen synthase activity was increased by as much as 10-fold, with concomitant increases (up to 5-fold) in the glycogen content. The uridine diphosphoglucose concentrations were markedly decreased, consistent with the increase in glycogen synthase activity. Levels of glycogen phosphorylase in these muscles increased (up to 3-fold), whereas the amount of the insulin-sensitive glucose transporter 4 either remained unchanged or decreased. The observation that increasing glycogen synthase enhances glycogen accumulation supports the conclusion that the activation of glycogen synthase, as well as glucose transport, contributes to the accumulation of glycogen in response to insulin in skeletal muscle.

Muscle protein synthesis by positron-emission tomography with l-[methyl-11C]methionine in adult humans

Existing methods for assessing protein synthetic rates (PSRs) in human skeletal muscle are invasive and do not readily provide information about individual muscle groups. Recent studies in canine skeletal muscle yielded PSRs similar to results of simultaneous stable isotope measurements using l-[1-13C, methyl-2H3]methionine, suggesting that positron-emission tomography (PET) with l-[methyl-11C]methionine could be used along with blood sampling and a kinetic model to provide a less invasive, regional assessment of PSR. We have extended and refined this method in an investigation with healthy volunteers studied in the postabsorptive state. They received ≈25 mCi of l-[methyl-11C]methionine with serial PET imaging of the thighs and arterial blood sampling for a period of 90 min. Tissue and metabolite-corrected arterial blood time activity curves were fitted to a three-compartment model. PSR (nmol methionine⋅min−1⋅g muscle tissue−1) was calculated from the fitted parameter values and the plasma methionine concentrations, assuming equal rates of protein synthesis and degradation. Pooled mean PSR for the anterior and posterior sites was 0.50 ± 0.040. When converted to a fractional synthesis rate for mixed proteins in muscle, assuming a protein-bound methionine content of muscle tissue, the value of 0.125 ± 0.01%⋅h−1 compares well with estimates from direct tracer incorporation studies, which generally range from ≈0.05 to 0.09%⋅h−1. We conclude that PET can be used to estimate skeletal muscle PSR in healthy human subjects and that it holds promise for future in vivo, noninvasive studies of the influences of physiological factors, pharmacological manipulations, and disease states on this important component of muscle protein turnover and balance.