Effects of the selective inhibition of proteasome caspase-like activity by CLi a derivative of nor-cerpegin in dystrophic mdx mice

Abstract

Previous studies have shown that proteasome inhibition can have beneficial effects in dystrophic mouse models. In this study, we have investigated the effects of a new selective proteasome inhibitor, CLi, a strong caspase-like inhibitor of the 20S proteasome, on skeletal and cardiac muscle functions of mdx mice. In the first series of experiments, five-month-old male mdx mice (n = 34) were treated with 2 different doses (20 and 100 μg/kg) of CLi and in the second series of experiments, five-month-old female mdx (n = 19) and wild-type (n = 24) mice were treated with 20 μg/kg CLi and Velcade (1 mg/kg) for 1-month. All animals were treadmill exercised twice a week to worsen the dystrophic features. In the first series of experiments, our results demonstrated that 20 μg/kg CLi did not significantly increase absolute and specific maximal forces in skeletal muscle from male mdx mice. Moreover, the higher susceptibility to contraction induced skeletal muscle injury was worsened by 100 μg/kg CLi since the force drop following lengthening contractions was increased with this high dose. Furthermore, we found no differences in the mRNA levels of the molecular markers implicated in dystrophic features. Concerning cardiac function, CLi had no effect on left ventricular function since ejection and shortening fractions were unchanged in male mdx mice. Similarly, CLi did not modify the expression of genes implicated in cardiac remodeling. In the second series of experiments, our results demonstrated an improvement in absolute and specific maximal forces by CLi, whereas Velcade only increased specific maximal force in female mdx mice. In addition, exercise tolerance was not improved by CLi. Taken together, our results show that CLi treatment can only improve maximal force production in exercised female mdx mice without affecting either exercice tolerance capacity or cardiac function. In conclusion, selective inhibition of caspase-like activity of proteasome with CLi has no compelling beneficial effect in dystrophic mdx mice.

Desmin prevents muscle wasting, exaggerated weakness and fragility, and fatigue in dystrophic mdx mouse

“…The protein concentration was measured using the Bradford method with bovine serum albumin as a standard. The proteasomal chymotrypsin-like, trypsin-like and caspase-like activities of the 20S catalytic core were assayed (Hovhannisyan et al 2019) using the fluorogenic substrates N-su ccinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC; Enzo Life Sciences, Villeurbanne, France), Bz-Val-Gly-Arg-7-amino-4-methylcoumarin (Bz-VGR-AMC; Enzo Life Sciences) and Z-Leu-Leu-Glu-7-amino-4-methylcoumarin (Z-LLE-AMC; Enzo Life Sciences), respectively. The mixture, containing 10 µg of total protein in 20 mM Tris (pH 8) and 10% glycerol, was incubated at 37°C with 20 µM peptide substrates in a final volume of 100 µL.…”

Treadmill running and mechanical overloading improved the strength of the plantaris muscle in the dystrophin‐desmin double knockout (DKO) mouse

“…Activin receptor administration increased absolute maximal force of the TA muscle AR increased absolute maximal force of TA muscle in DKO+AR mice, but it remained lower (mean: 400 mN) than observed in adult healthy mice (means: 1120, 1190 and 1270 mN) (Ferry et al, 2020;Hovhannisyan et al, 2019;Roy et al, 2016) (Fig. 7E).…”

Absence of Desmin Results in Impaired Adaptive Response to Mechanical Overloading of Skeletal Muscle

“…Tissue homogenates containing proteasome were prepared just after dissection using ice-cold homogenization buffer containing: 20 mM Tris-HCl (pH 7.6), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA and 2 mM DTT (Hovhannisyan et al, 2019). Samples were minced with scissors and homogenized using plastic pestles, incubated 30 min on ice, then centrifuged at 12,000 g for 15 min at 4 • C. Protein concentration was measured using the Bradford method with bovine serum albumin as a standard.…”