Pharyngeal pathology in a mouse model of oculopharyngeal muscular dystrophy is associated with impaired basal autophagy in myoblasts

Abstract

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset dominant disease that primarily affects craniofacial muscles. Despite the fact that the genetic cause of OPMD is known to be expansion mutations in the gene encoding the nuclear polyadenosine RNA binding protein PABPN1, the molecular mechanisms of pathology are unknown and no pharmacologic treatments are available. Due to the limited availability of patient tissues, several animal models have been employed to study the pathology of OPMD. However, none of these models have demonstrated functional deficits in the muscles of the pharynx, which are predominantly affected by OPMD. Here, we used a knock-in mouse model of OPMD, Pabpn1+/A17, that closely genocopies patients. In Pabpn1+/A17 mice, we detected impaired pharyngeal muscle function, and impaired pharyngeal satellite cell proliferation and fusion. Molecular studies revealed that basal autophagy, which is required for normal satellite cell function, is higher in pharynx-derived myoblasts than in myoblasts derived from limb muscles. Interestingly, basal autophagy is impaired in cells derived from Pabpn1+/A17 mice. Pabpn1 knockdown in pharyngeal myoblasts failed to recapitulate the autophagy defect detected in Pabpn1+/A17 myoblasts suggesting that loss of PABPN1 function does not contribute to the basal autophagy defect. Taken together, these studies provide the first evidence for pharyngeal muscle and satellite cell pathology in a mouse model of OPMD and suggest that aberrant gain of PABPN1 function contributes to the craniofacial pathology in OPMD.

Regulation of Atp7a RNA contributes to differentiation-dependent Cu redistribution in skeletal muscle cells

“…Specific proteins were quantified by immunoblot as previously described. 57 Cells were washed twice in PBS and then incubated in radioimmunoprecipitation buffer [10 mM piperazine- N - N -bis(2-ethanesulfonic acid)] supplemented with protease inhibitor (Pierce) and phosphatase inhibitor (Sigma Aldrich). Samples were sonicated on ice three times, 10 s each at ∼40% output on an ultrasonic membrane disruptor (Fisher Model 100).…”

“…Steady-state transcript levels were determined using quantitative PCR on reverse-transcribed cDNAs (RT-qPCR) as previously described. 57 RNA was isolated using TriZol (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized from 500 ng of RNA using the Maxima First Strand cDNA Synthesis Kit with dsDnase (ThermoFisher).…”

“…Primary muscle stem cell-derived myoblasts were isolated from hindlimb muscles of five combined male Atp7a fl/y mice using magnetic cell sorting as previously described. 57 Muscle tissue was rinsed in PBS, minced, and then digested in DMEM with 0.1% Pronase (EMD Millipore) and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4 for 1 h at 37°C with gentle agitation. Digests were centrifuged and resuspended in DMEM containing 10% FBS and pen/strep, triturated using a 25 ml serological pipette and insoluble material was separated using 100 µM Steriflip filters (EMD Millipore).…”