Targeting the Ubiquitin-Proteasome System in Limb-Girdle Muscular Dystrophy With CAPN3 Mutations

Abstract

LGMDR1 is caused by mutations in the CAPN3 gene that encodes calpain 3 (CAPN3), a non-lysosomal cysteine protease necessary for proper muscle function. Our previous findings show that CAPN3 deficiency leads to reduced SERCA levels through increased protein degradation. This work investigates the potential contribution of the ubiquitin-proteasome pathway to increased SERCA degradation in LGMDR1. Consistent with our previous results, we observed that CAPN3-deficient human myotubes exhibit reduced SERCA protein levels and high cytosolic calcium concentration. Treatment with the proteasome inhibitor bortezomib (Velcade) increased SERCA2 protein levels and normalized intracellular calcium levels in CAPN3-deficient myotubes. Moreover, bortezomib was able to recover mutated CAPN3 protein in a patient carrying R289W and R546L missense mutations. We found that CAPN3 knockout mice (C3KO) presented SERCA deficits in skeletal muscle in the early stages of the disease, prior to the manifestation of muscle deficits. However, treatment with bortezomib (0.8 mg/kg every 72 h) for 3 weeks did not rescue SERCA levels. No change in muscle proteasome activity was observed in bortezomib-treated animals, suggesting that higher bortezomib doses are needed to rescue SERCA levels in this model. Overall, our results lay the foundation for exploring inhibition of the ubiquitin-proteasome as a new therapeutic target to treat LGMDR1 patients. Moreover, patients carrying missense mutations in CAPN3 and presumably other genes may benefit from proteasome inhibition by rescuing mutant protein levels. Further studies in suitable models will be necessary to demonstrate the therapeutic efficacy of proteasome inhibition for different missense mutations.

A knock down strategy for rapid, generic, and versatile modelling of muscular dystrophies in 3D-tissue-engineered-skeletal muscle

“…The 3D-TESMs recapitulate these abnormalities, as we observed a dysregulation of several ankyrin proteins (ANK2 and ANK3), ankyrin repeat proteins (ANKRD1, ANKRD2, and ANKS1A), and catenin (CTNNA1, CTNNB1, and CTNND1) isoforms which provide structural integrity to the cytoskeleton and are known substrates of Calpain-3. In addition, we observed a downregulation of multiple PSMA and PSMB isoforms, which compose the active site of the 20S proteasome, resembling previous findings made in Calpain-3-deficient (C3KO) mice and immortalized human CAPN3-deficient cell lines by [ 36 ].…”

“…Using mass spectrometry to obtain proteomic profiles, distinct proteomic signatures were obtained, corresponding with previous studies performed with patient biopsies or animal models of Duchenne muscular dystrophy and LGMD2A. Calpain-3 is a calcium-dependent protease responsible for the cleavage of cytoskeletal proteins and actin filaments in skeletal muscle; it is therefore a prerequisite for the maintenance and remodeling of healthy skeletal muscle [ 23 , 35 , 36 ]. While the exact molecular mechanisms underlying the pathology of LGMD2A remain unclear, CAPN3-deficient muscles have been found to exhibit structural abnormalities of the sarcomere and cytoskeleton [ 22 , 23 , 37 , 38 ].…”

Limb-girdle muscular dystrophies: An update

“…Other drugs which have been used without supportive trial evidence include rituximab, ubiquitin-proteasome inhibitors i.e., bortezomib, givinostat, coenzyme Q and lisinopril. [40][41][42] ⏐ SAMARASIRI SLJoN…”

Skeletal muscle cells derived from induced pluripotent stem cells: A platform for limb girdle muscular dystrophies

“…To overcome these issues, several in vitro models were developed through transdifferentiation of non-muscle cells into skeletal cells by forced expression of myogenic transcription factors 21 . More recently, immortalization of primary cells with hTERT and CDK4 was reported as successful for modeling LGMD 22,23 , including LGMDD4/R1 24 , LGMDR2 25–27 and LGMDR12 28 While these different models present several advantages in terms of proliferation and maturity, the development of transgene-free cellular models from non-invasive procedures was proposed through the use of pluripotent stem cells.…”