Molecular and Cellular Pharmacology
Comparison between gentamycin and exon skipping treatments to restore ryanodine receptor subtype 2 functions in mdx mouse duodenum myocytes

https://doi.org/10.1016/j.ejphar.2009.11.034Get rights and content

Abstract

In Duchenne muscular dystrophy, a stop-codon mutation in the dystrophin gene induces an impairment of skeletal and smooth muscles contraction. In duodenum from mdx mouse, the disease model, the decrease of contractility was linked with the decrease of calcium signals encoded by ryanodine receptor subtype 2. Aminoglycoside and antisense oligonucleotide strategies were investigated to restore calcium signalling in the mdx mouse. Mdx mice were treated by intraperitoneal injection of gentamycin or 2-O-methyl antisense ribonucleotide directed against exon 23 of dystrophin for 2 weeks. The efficiency of both therapeutic strategies was determined by the level of dystrophin protein expression. The physiological effects of both treatments on ryanodine receptor expression and function were followed by RT-PCR, western blot and calcium measurements. Fourteen days after injection of gentamycin or anti-dystrophin antisense, the expression of dystrophin was recovered in skeletal muscle from treated mdx mice. In duodenum cells, RT-PCR and western blot indicated that the expression of ryanodine receptor subtype 2 was similar in treated mice than in control mice in association with the recovery of caffeine-induced Ca2+ response. No significant difference was observed in the ryanodine subtype 3-dependent spontaneous Ca2+ oscillations in untreated and treated mice. Conclusions — these results may help to explain the efficiency of aminoglycoside and anti-dystrophin antisense treatments in smooth muscle. Both treatments could be an interesting therapeutic option to restore smooth muscle contraction in patients with Duchenne muscular dystrophy.

Introduction

Dystrophin is a cytoskeletal structural protein ubiquitously expressed. Its absence causes Duchenne muscular dystrophy. In the mdx mouse (Duchenne muscular dystrophy murine model), the lack of dystrophin is due to a punctual mutation introducing a stop codon in exon 23 (Sicinski et al., 1989). Dysfunctions of smooth muscles were also observed and induced severe pathologies such as a decrease of intestinal peristaltsis (Leon et al., 1986). In human and mdx mice skeletal muscle, the absence of dystrophin induces the alteration of plasma membrane integrity in association with Ca2+ homeostasis impairment (Turner et al., 1988).

The excitation–contraction coupling is under control of the Ca2+ signalling and the release of stored Ca2+ from the sarcoplasmic reticulum to the cytosol is implicated in Ca2+-dependent contraction. The ryanodine-sensitive Ca2+ release channel ( ryanodine receptor) encodes the Ca2+-induced Ca2+ release (CICR) mechanism, which appears to be the dominant process for the release of Ca2+ from the sarcoplasmic reticulum via amplification of Ca2+ influxes and inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release to induce contraction (Morel et al., 2007). Three different subtypes of ryanodine receptors were cloned and named RYR1, RYR2 and RYR3. In duodenum myocytes, RYR2 and two different isoforms of RYR3 are expressed. The RYR2 subtype is necessary to encode the CICR and its activity is negatively regulated by the short isoform of RYR3 (Dabertrand et al., 2006).

The absence of dystrophin in skeletal as well as smooth muscle has been correlated with the dysfunction of the ryanodine receptor-dependent Ca2+ signalling. In skeletal muscle, the coupling between RYR1 and voltage-dependent Ca2+ channel was damaged (Wang et al., 2005) whereas in duodenum the decrease of CICR efficiency was linked to the decrease of RYR2 expression (Morel et al., 2004). Although the disorderly changes in resting Ca2+ concentration and spontaneous Ca2+ signals were found in skeletal muscle, in smooth muscle there was no evidence that they were perturbed drastically.

Aminoglycoside antibiotics such as gentamycin have been shown to suppress the nonsense mutation in vivo and can thus restore, in mdx, the muscular functions in skeletal and smooth muscle (Barton-Davies et al., 1999; Loufrani et al., 2004). The middle part of the dystrophin protein contained repeated domains encoded by contiguous exons potentially containing a mutation in the dystrophin gene responsible for the observed dystrophic phenotype. Thus deletion of the exon containing the mutation responsible for dystrophy could also be accomplished via an antisense oligonucleotide strategy. This strategy was named “exon skipping.” In the mdx mouse, the systemic delivery of an antisense oligonucleotide directed against exon 23 induced the expression of a truncated form of dystrophin (Lu et al., 2003). However, whereas the restoration of smooth muscle functions by gentamycin was demonstrated (Loufrani et al., 2004), the effect of expression of the truncated form of dystrophin by inducing exon skipping by antisense oligonucleotide was not studied in smooth muscle.

Here, we investigated whether in mdx mice, gentamycin and anti-dystrophin antisense oligonucleotide treatments could restore: (1) both dystrophin and RYR2 expressions and (2) ryanodine receptor-dependent Ca2+ release.

Section snippets

Gentamycin treatment

The mdx mice were injected intra-peritoneally with gentamycin sulphate (MPBiomedicals, ref 194530, batch no. R16139) or “saline” solution daily (34 µg/g per injection in 0.4 ml of 5% glucose solution (Loufrani et al., 2004). Mice were euthanized after 14 days of treatment.

Anti-dystrophin antisense RNA treatment

The 2-O-methyl antisense phosphorothioate oligoribonucleotide directed against exon 23 of mouse dystrophin mRNA sequence (asDYS: 5′-GGCCAAACCUCGGCUUACCU-3′) (Lu et al., 2003) was synthesized tagged with the Cy5 fluorophore

Effect of gentamycin and asDYS treatments on RYR2 expression

First, we verified the efficiency of gentamycin and asDYS treatments by western blot analysis of expression of dystrophin on thigh skeletal muscle from each treated animal. The efficiency of asDYS to induce the skipping of exon 23 was verified in duodenum myocytes by RT-PCR (Fig. S1). We have also verified the recovery of dystrophin expression by pharmacological treatments in duodenum smooth muscle. The western blot of dystrophin was performed with proteins extracted from duodenum smooth muscle

Discussion

In patients with Duchenne muscular dystrophy as in mdx mice, it is generally accepted that the missing link between the absence of dystrophin and muscle hypotonia is due to alterations in Ca2+ homeostasis. In all muscle types of the mdx model, the ryanodine receptor subtypes could be implicated. In skeletal muscle, the RYR1-dependent Ca2+ sparks were disorganized without significant modification of RYR1 expression (Wang et al., 2005, Bellinger et al., 2009); in cardiac myocytes, the decrease of

Acknowledgments

Grants were from the Association Française contre les myopathies and Centre National de la Recherche Scientifique (CNRS). The authors want to thank Dr. Anne Prevot for the proofreading and N. Biendon and N. Henkous for the RT-PCR experiments. JLM is the principal investigator for this project. With FD, they have treated the mice, prepared the samples and made the calcium experiments. MH made the western blot experiments. NM and JM were implicated in the critical reading of the manuscript.

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