Calpain activation impairs neuromuscular transmission in a mouse model of the slow-channel myasthenic syndrome
Jason S. Groshong, … , Richard J. Miller, Christopher M. Gomez
Jason S. Groshong, … , Richard J. Miller, Christopher M. Gomez
Published October 1, 2007
Citation Information: J Clin Invest. 2007;117(10):2903-2912. https://doi.org/10.1172/JCI30383.
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Calpain activation impairs neuromuscular transmission in a mouse model of the slow-channel myasthenic syndrome

Abstract

The slow-channel myasthenic syndrome (SCS) is a hereditary disorder of the acetylcholine receptor (AChR) of the neuromuscular junction (NMJ) that leads to prolonged AChR channel opening, Ca2+ overload, and degeneration of the NMJ. We used an SCS transgenic mouse model to investigate the role of the calcium-activated protease calpain in the pathogenesis of synaptic dysfunction in SCS. Cleavage of a fluorogenic calpain substrate was increased at the NMJ of dissociated muscle fibers. Inhibition of calpain using a calpastatin (CS) transgene improved strength and neuromuscular transmission. CS caused a 2-fold increase in the frequency of miniature endplate currents (MEPCs) and an increase in NMJ size, but MEPC amplitudes remained reduced. Persistent degeneration of the NMJ was associated with localized activation of the non-calpain protease caspase-3. This study suggests that calpain may act presynaptically to impair NMJ function in SCS but further reveals a role for other cysteine proteases whose inhibition may be of additional therapeutic benefit in SCS and other excitotoxic disorders.

Authors

Jason S. Groshong, Melissa J. Spencer, Bula J. Bhattacharyya, Elena Kudryashova, Bhupinder P.S. Vohra, Roberto Zayas, Robert L. Wollmann, Richard J. Miller, Christopher M. Gomez

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Figure 1

Calpain activity is elevated in transgenic SCS muscle fibers.

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Calpain activity is elevated in transgenic SCS muscle fibers. (A and B) ...
(A and B) Representative images of acutely dissociated εL269F transgenic (A) and WT (B) FDB muscle fibers after loading with 10 μM BOC-Leu-Met-CMAC and labeling endplates (white arrowheads) with RαBT (pseudocolored pink). (C) Average fluorescence intensity for εL269F transgenic or WT BOC-Leu-Met-CMAC–loaded muscle fibers after no treatment (Un) or treatment with vehicle (Vh) or peptide calpain inhibitor (CP1). Calpain activity of εL269F transgenic muscle fibers was significantly elevated when compared with that of WT (1,591 ± 72 vs. 815 ± 72; n = 10; P < 0.01). No substantial effect was observed for either genotype when the fibers were treated with vehicle (εL269F: 1,591 ± 72, n = 10, vs. 1,593 ± 72, n = 3, and WT: 815.1 ± 72, n = 10, vs. 855.3 ± 52, n = 3; P > 0.05). CP1 pretreatment of εL269F muscle fibers significantly reduced calpain activity (vehicle: 1,591 ± 72, n = 10, vs. CP1: 801.6 ± 25, n = 3; P < 0.01), with values similar to those obtained for WT animals (P > 0.05). Calpain activity of CP1-treated WT fibers was similar to that of untreated WT fibers (924 ± 34, n = 3, vs. 815.1 ± 72, n = 10; P > 0.05). Scale bar: 100 μm.