Molecular and Cellular PharmacologyComparison between gentamycin and exon skipping treatments to restore ryanodine receptor subtype 2 functions in mdx mouse duodenum myocytes
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.
References (25)
- et al.
Modulation of calcium signalling by dominant negative splice variant of ryanodine receptor subtype 3 in native smooth muscle cells
Cell Calcium
(2006) - et al.
Full length ryanodine receptor subtype 3 encodes spontaneous calcium oscillations in native duodenal smooth muscle cells
Cell Calcium
(2008) - et al.
Morpholino oligomer-mediated exon skipping averts the onset of dystrophic pathology in the mdx mouse
Mol. Ther.
(2007) - et al.
Chronic intestinal pseudoobstruction as a complication of Duchenne's muscular dystrophy
Gastroenterology
(1986) - et al.
Decreased mAKAP, ryanodine receptor, and SERCA2a gene expression in mdx hearts
Biochem. Biophys. Res. Commun.
(2003) - et al.
Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice
J. Clin. Invest.
(1999) - et al.
Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle
Nat. Med.
(2009) - et al.
Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment
Gene Ther.
(2004) - et al.
Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide
J. Gene Med.
(2006) - et al.
Ryanodine receptor subtype 2 encodes Ca2+ oscillations activated by acetylcholine via the M2 muscarinic receptor/cADP-ribose signalling pathway in duodenum myocytes
J. Cell Sci.
(2005)
Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations
Nat. Med.
Absence of dystrophin in mice reduces NO-dependent vascular function and vascular density: total recovery after a treatment with the aminoglycoside gentamicin
Arterioscler. Thromb. Vasc. Biol.
Cited by (7)
Hydroxysafflor yellow A actives BK<inf>Ca</inf> channels and inhibits L-type Ca channels to induce vascular relaxation
2020, European Journal of PharmacologyCitation Excerpt :We found that HSYA had an obviously inhibitory effect on Ca-L channels in whole-cell recording mode, which has not been reported so far. It was linked to the decrease of calcium influx and might further lead to the inactivation of downstream pathways (Dabertrand et al., 2010), which required further research. This indicates that the inhibition of ICa-L might contribute to the vasodilatory action of HSYA.
Blood brain barrier precludes the cerebral arteries to intravenously-injected antisense oligonucleotide.
2015, European Journal of PharmacologyCitation Excerpt :If some molecules passed through the wall vessels, the nature of the molecule could be important. That is why we compared the diffusion of asRyR3S and asDYS that we had previously used in vivo to target smooth muscle cells of portal vein and duodenum (Dabertrand et al., 2010; Morel et al., 2009). As illustrated in the center and the right of Fig. 6, the presence of asDYS was more important than the presence of asRyR3 in liver and heart, but in brain both asDYS and asRyR3S were not enough detected.
Aminoglycoside modifying enzymes
2010, Drug Resistance UpdatesCitation Excerpt :The property of aminoglycosides to decrease the fidelity of the eukaryotic elongation machinery makes them potential candidates to treat nonsense mutation related genetic disorders such as those mentioned above or others that can benefit from inducing translational readthrough (Hermann, 2007; Kellermayer, 2006; Zingman et al., 2007). Aminoglycosides, mainly gentamicin, have also been used in the treatment of Ménière's disease by intratympanic injection (Dabertrand et al., 2010; Nakashima et al., 2000). Aminoglycoside-based drugs are also inhibitors of reproduction of the HIV virus, showing promise on the treatment of AIDS (reviewed in Houghton et al., 2010).
Ongoing therapeutic trials and outcome measures for Duchenne muscular dystrophy
2013, Cellular and Molecular Life SciencesSpaceflight regulates ryanodine receptor subtype 1 in portal vein myocytes in the opposite way of hypertension
2012, Journal of Applied Physiology