Dystrophin Threshold Level Necessary for Normalization of Neuronal Nitric Oxide Synthase, Inducible Nitric Oxide Synthase, and Ryanodine Receptor-Calcium Release Channel Type 1 Nitrosylation in Golden Retriever Muscular Dystrophy Dystrophinopathy.
At present, the clinically most advanced strategy to treat Duchenne muscular dystrophy (DMD) is the exon-skipping strategy. Whereas antisense oligonucleotide-based clinical trials are underway for DMD, it is essential to determine the dystrophin restoration threshold needed to ensure improvement of muscle physiology at the molecular level. A preclinical trial has been conducted in golden retriever muscular dystrophy (GRMD) dogs treated in a forelimb by locoregional delivery of rAAV8-U7snRNA to promote exon skipping on the canine dystrophin messenger. Here, we exploited rAAV8-U7snRNA-transduced GRMD muscle samples, well characterized for their percentage of dystrophin-positive fibers, with the aim of defining the threshold of dystrophin rescue necessary for normalization of the status of neuronal nitric oxide synthase mu (nNOSμ), inducible nitric oxide synthase (iNOS), and ryanodine receptor-calcium release channel type 1 (RyR1), crucial actors for efficient contractile function. Results showed that restoration of dystrophin in 40% of muscle fibers is needed to decrease abnormal cytosolic nNOSμ expression and to reduce overexpression of iNOS, these two parameters leading to a reduction in the NO level in the muscle fibers. Furthermore, the same percentage of dystrophin-positive fibers of 40% was associated with the normalization of RyR1 nitrosylation status and with stabilization of the RyR1-calstabin1 complex that is required to facilitate coupled gating. We concluded that a minimal threshold of 40% of dystrophin-positive fibers is necessary for the reinstatement of central proteins needed for proper muscle contractile function, and thus identified a rate of dystrophin expression significantly improving, at the molecular level, the dystrophic muscle physiology.
1 Sorbonne Universités , UPMC Univ Paris 06/INSERM/CNRS/Institut de Myologie/Centre de Recherche en Myologie (CRM), GH Pitié Salpêtrière, Paris, France.
Christel
C
Gentil
2 Atlantic Gene Therapies/INSERM UMR 1089 Université de Nantes , CHU de Nantes, IRT1, Nantes, France.|3 Généthon , Evry, France.
Caroline
C
Le Guiner
1 Sorbonne Universités , UPMC Univ Paris 06/INSERM/CNRS/Institut de Myologie/Centre de Recherche en Myologie (CRM), GH Pitié Salpêtrière, Paris, France.
Sestina
S
Falcone
4 Institut de Myologie , GH Pitié-Salpêtrière, Paris, France.
Jean-Yves
JY
Hogrel
1 Sorbonne Universités , UPMC Univ Paris 06/INSERM/CNRS/Institut de Myologie/Centre de Recherche en Myologie (CRM), GH Pitié Salpêtrière, Paris, France.
Cécile
C
Peccate
1 Sorbonne Universités , UPMC Univ Paris 06/INSERM/CNRS/Institut de Myologie/Centre de Recherche en Myologie (CRM), GH Pitié Salpêtrière, Paris, France.
Stéphanie
S
Lorain
1 Sorbonne Universités , UPMC Univ Paris 06/INSERM/CNRS/Institut de Myologie/Centre de Recherche en Myologie (CRM), GH Pitié Salpêtrière, Paris, France.
Sofia
S
Benkhelifa-Ziyyat
5 Atlantic Gene Therapies /INRA UMR 703, ONIRIS, Nantes-Atlantic National College of Veterinary Medicine , Food Science, and Engineering, Nantes, France .
Lydie
L
Guigand
3 Généthon , Evry, France.
Marie
M
Montus
4 Institut de Myologie , GH Pitié-Salpêtrière, Paris, France.
Laurent
L
Servais
1 Sorbonne Universités , UPMC Univ Paris 06/INSERM/CNRS/Institut de Myologie/Centre de Recherche en Myologie (CRM), GH Pitié Salpêtrière, Paris, France.
Thomas
T
Voit
1 Sorbonne Universités , UPMC Univ Paris 06/INSERM/CNRS/Institut de Myologie/Centre de Recherche en Myologie (CRM), GH Pitié Salpêtrière, Paris, France.
France
F
Piétri-Rouxel
D000818:Animals; D004285:Dogs; D016189:Dystrophin / Q000378:metabolism*; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D015538:Nitrosation; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
D016189:Dystrophin; D019837:Ryanodine Receptor Calcium Release Channel; D009569:Nitric Oxide; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II