Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice

Abstract

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene, leading to the absence of the dystrophin protein in striated muscle. A significant number of these mutations are premature stop codons. On the basis of the observation that aminoglycoside treatment can suppress stop codons in cultured cells, we tested the effect of gentamicin on cultured muscle cells from the mdx mouse - an animal model for DMD that possesses a premature stop codon in the dystrophin gene. Exposure of mdx myotubes to gentamicin led to the expression and localization of dystrophin to the cell membrane. We then evaluated the effects of differing dosages of gentamicin on expression and functional protection of the muscles of mdx mice. We identified a treatment regimen that resulted in the presence of dystrophin in the cell membrane in all striated muscles examined and that provided functional protection against muscular injury. To our knowledge, our results are the first to demonstrate that aminoglycosides can suppress stop codons not only in vitro but also in vivo. Furthermore, these results raise the possibility of a novel treatment regimen for muscular dystrophy and other diseases caused by premature stop codon mutations. This treatment could prove effective in up to 15% of patients with DMD.

Figure 1

Immunohistochemistry of myotubes from primary cell culture from C57 and mdx muscle. The presence of dystrophin was detected by mAb to the COOH-terminus of dystrophin (F192A12) followed by a rhodamine-conjugated anti-mouse IgG. Dystrophin was present in myotubes from C57 mice (left) and in mdx myotubes treated with 300 μg/mL (middle). No dystrophin was detected in untreated mdx myotubes (right).

Figure 2

The effect of gentamicin treatment on eccentric contraction injury. Effective treatment regimes are depicted in a. The decrement in force after 5 eccentric contractions was significantly less in mdx EDL muscles injected with gentamicin (mdxGi200, n = 8; 200% dose equivalent) compared with untreated mdx EDLs (mdxctrl, n = 8) and muscles from Becker construct (22) transgenic animals (Beck tg, n = 5). The level of protection afforded by gentamicin in mdx muscles approached the values measured in EDLs from untreated C57 mice (C57ctrl, n = 4), and was not significantly different from EDLs from gentamicin-treated C57 mice (C57Gi200, n = 4). DHB treatment, when combined with 200% gentamicin (mdxGi200D, n = 4), did not alter levels of protection by this gentamicin dose alone. Lower injected doses of gentamicin (50% dose equivalent) were very effective at preventing force decrements in female mdx mice (mdxGi50F, n = 4). Treatment regimens that were not effective in preventing eccentric contractile injury in male mdx mice are displayed in b. These include 400% gentamicin injected in combination with DHB (mdxGi400D, n = 4), 100% injected gentamicin (mdxGi100, n = 4), and all mdx animals treated by infusion pumps (mdxGP50, 50% dose; mdxGP100, 100% dose; mdxGP200, 200% dose; n = 4 for each condition). The measurements of force generation after eccentric contraction were reflected in the measurements of membrane damage (c). There was a significant reduction in the proportion of fibers with dye entry in EDLs from treated mdx mice compared with muscles from untreated mdx muscles. P < 0.05 for comparisons between treated mdx muscles and untreated C57 controls. *P < 0.05 for comparisons between treated and untreated mdx muscles.

Figure 3

Immunohistochemistry of muscle cross-sections to view dystrophin (a–c) and γ-sarcoglycan (d–f). C57 control TA displayed positive staining for both proteins (a and d). Lower staining intensity for dystrophin and γ-sarcoglycan was observed in mdx TAs treated with gentamicin (b and e). No dystrophin staining, and only minimal staining of γ-sarcoglycan, were seen in mdx control TA muscles (c and f). Scale bar: 25 μm.

Figure 4

Immunoblot analysis of TA muscles for dystrophin and γ-sarcoglycan. Gentamicin treatment at the 200% dose equivalent significantly increased both dystrophin and γ-sarcoglycan levels above those in untreated mdx muscle (lane 2) when introduced by injection (lane 3) or by infusion pump (lane 5) into male mdx mice. The protein levels were approximately 10–20% those of C57 controls (lane 1). Female mdx mice treated with only 50% doses of gentamicin (lane 4) also displayed significant levels of both proteins compared with untreated controls.

Figure 5

The effect of gentamicin treatment on serum CK levels. The levels of serum CK in untreated mdx mice (n = 4) were significantly reduced in mdx mice treated with 200% gentamicin by daily injection (n = 4). These levels approached those measured in C57 control mice (n = 2). P < 0.05 for comparisons between treated mdx muscles and untreated C57 controls. *P < 0.05 for comparisons between treated and untreated mdx muscles.