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.
Highlights
Limb-girdle muscular dystrophy recessive 1 (LGMDR1), or calpainopathy, is the most common form of limb-girdle dystrophies
We reported reduced sarco/endoplasmic reticulum calcium ATPase (SERCA) protein levels together with diminished SERCA ATPase activity in human (LHCN-M2) and mouse (C2C12) myotubes knockdown for calpain 3 (CAPN3) (Toral-Ojeda et al, 2016)
At the mRNA level, we found that CAPN3 deficiency does not affect ATP2A1 and ATP2A2 expression, codifying for SERCA1 and SERCA2, respectively
Summary
Limb-girdle muscular dystrophy recessive 1 (LGMDR1), or calpainopathy (previously known as LGMD2A), is the most common form of limb-girdle dystrophies. It is caused by mutations in the CAPN3 gene, which encodes the proteolytic enzyme calpain 3 (CAPN3), a non-lysosomal cysteine protease (Richard et al, 1995). This disease is characterized by progressive degeneration of scapular. Most therapeutic strategies for LGMDR1 are focusing on correcting the primary genetic defect through gene or cell therapies (Roudaut et al, 2013; Straub and Bertoli, 2015; Lasa-Elgarresta et al, 2019; Selvaraj et al, 2019; Sahenk et al, 2021). There is a need for developing novel pharmacological therapies directed towards alternative targets of the disease
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