Investigation of Functional Impact of Fgfr1 Missplicing on Skeletal Muscle of Myotonic Dystrophy

Project: National Science and Technology CouncilNational Science and Technology Council Academic Grants

Project Details


Myotonic dystrophy (DM) is the most common adult onset muscular dystrophy with multisystemic features. Skeletal muscle symptoms including myotonia, weakness and atrophy are the hallmark of this disease. Among those, muscle atrophy is the major concern of patients and may cause respiratory failure. However, the molecular mechanisms underlying the most common cause of death in DM are yet to be defined. DM is caused by abnormal expansion of microsatellite (C(C)TG)n in the 3’-UTR of the DMPK gene (DM type 1, DM1) and the first intron of CNBP gene (DM type 2, DM2). The transcribed (C(C)UG)n RNAs form double-stranded hairpins and sequester Muscleblind-like (MBNL) family proteins that is critical for fetal to adult transition. The sequestrations lead to fetal isoform retention in adult tissues that account for various “fetus-like” symptoms in DM. Recently, we successfully created Mbnl1 knockout (KO) and Mbnl2 KO mice, which reproduce skeletal muscle and CNS symptoms, respectively. We crossed these two lines and created Mbnl1ΔE3/ΔE3; Mbnl2ΔE2/+ (1KO2HET) and Mbnl1ΔE3/ΔE3; Mbnl2flox/flox; Myo-Cre+/- (Myo-Cre DKO) mice representing the only rodent models with comprehensive DM skeletal muscle phenotypes. Although we have demonstrated extensive splicing misregulations in these mouse models, the potential contributions of MBNL-regulated downstream targets to muscle wasting remain elusive. To establish the link between splicing misregulation and muscular atrophy in DM, we choose to investigate the functional impact of FGFR1 missplicing, a key tyrosine kinase receptor for a variety of FGFs. Our specific aims are: Aim 1. To explore the effects of MBNL dysfunction-induced FGFR1 missplicing on myogenesis in the mouse model of DM Mouse myoblast C2C12 cells will be cultured using proliferating and differentiating medium to induce myogenesis. Firstly, transfection experiments using plasmids expressing two Fgfr1 isoforms will be performed. Secondly, the antisense oligonucleotide (AON) will be transfected into cells and modulate Fgfr1 splicing. Thirdly, we will evaluate the rescuing effect of intramuscular-injected AON for the regeneration after notexin proteolysis in the muscle of 1KO2HET mice. Aim 2. To examine the influences of FGFR1 splicing misregulation on satellite cell homeostasis. The number and function of satellite cells (SCs) in 1KO2HET mice will be investigated by immunostaining, fluorescence-activated cell sorting (FACS) and colonal myogenesis assay. The impact of splicing modifying AON on SCs will also be evaluated. Aim 3. To investigate the therapeutic potential of AAV-mediated AON delivery of Fgfr1 in the skeletal muscle of DM mouse model Intramuscular injection of AAV-mediated AON will be evaluated for the long term effects of FGFR1 splicing and the capacity of rescuing pathophysiological function in 1KO2HET mice. Aim 4. To test the synergistic effects of combined use of FGF2 supplement and correction of FGFR1 missplicing for the muscle wasting in DM mouse model The supplement of FGF2 will be given to Myo-Cre DKO mice intramuscularly for the rescue of muscle atrophy. The effect of combined use of AON for modifying FGFR1 splicing will be evaluated.

Project IDs

Project ID:PC10608-1486
External Project ID:MOST106-2314-B182-030
Effective start/end date01/08/1731/07/18


Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.