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Effect of calpain and proteasome inhibition on Ca²⁺-dependent proteolysis and muscle histopathology in the mdx mouse

Santhera Pharmaceuticals, Liestal, Switzerland

¹Correspondence: Santhera Pharmaceuticals (Switzerland) Ltd, Hammerstrasse 47, CH-4410 Liestal, Switzerland. E-mail: guenther.metz{at}santhera.com

Dystrophin deficiency is the underlying molecular cause of progressive muscle weakness observed in Duchenne muscular dystrophy (DMD). Loss of functional dystrophin leads to elevated levels of intracellular Ca²⁺, a key step in the cellular pathology of DMD. The cysteine protease calpain is activated in dystrophin-deficient muscle, and its inhibition is regarded as a potential therapeutic approach. In addition, previous work has shown that the ubiquitin-proteasome system also contributes to muscle protein breakdown in dystrophic muscle and, therefore, also qualifies as a potential target for therapeutic intervention in DMD. The relative contribution of calpain- and proteasome-mediated proteolysis induced by increased Ca²⁺ levels was characterized in cultured muscle cells and revealed initial Ca²⁺ influx-dependent calpain activity and subsequent Ca²⁺-independent activity of the ubiquitin-proteasome system. We then set out to optimize novel small-molecule inhibitors that inhibit both calpain as well as the 20S proteasome in a cellular system with impaired Ca²⁺ homeostasis. On administration of such inhibitors to mdx mice, quantitative histological parameters improved significantly, in particular with compounds strongly inhibiting the 20S proteasome. To investigate the role of calpain inhibition without interfering with the ubiquitin-proteasome system, we crossed mdx mice with transgenic mice, overexpressing the endogenous calpain inhibitor calpastatin. Although our data show that proteolysis by calpain is strongly inhibited in the transgenic mdx mouse, this calpain inhibition did not ameliorate muscle histology. Our results indicate that inhibition of the proteasome rather than calpain is required for histological improvement of dystrophin-deficient muscle. In conclusion, we have identified novel proteasome inhibitors that qualify as potential candidates for pharmacological intervention in muscular dystrophy.—Briguet, A., Erb, M., Courdier-Fruh, I., Barzaghi, P., Santos, G., Herzner, H., Lescop, C., Siendt, H., Henneboehle, M., Weyermann, P., Magyar, J. P., Dubach-Powell, J., Metz, G., Meier, T. Effect of calpain and proteasome inhibition on Ca²⁺-dependent proteolysis and muscle histopathology in the mdx mouse.

Key Words: Duchenne muscular dystrophy • dystrophin deficiency • small-molecule inhibitors • calpastatin • in vivo efficacy