Leupeptin-based inhibitors do not improve the mdx phenotype

Abstract

Calpain activation has been implicated in the disease pathology of Duchenne muscular dystrophy. Inhibition of calpain has been proposed as a promising therapeutic target, which could lessen the protein degradation and prevent progressive fibrosis. At the same time, there are conflicting reports as to whether elevation of calpastatin, an endogenous calpain inhibitor, alters pathology. We compared the effects of pharmacological calpain inhibition in the mdx mouse using leupeptin and a proprietary compound (C101) that linked the inhibitory portion of leupeptin to carnitine (to increase uptake into muscle). Administration of C101 for 4 wk did not improve muscle histology, function, or serum creatine kinase levels in mdx mice. Mdx mice injected daily with leupeptin (36 mg/kg) for 6 mo also failed to show improved muscle function, histology, or creatine kinase levels. Biochemical analysis revealed that leupeptin administration caused an increase in m-calpain autolysis and proteasome activity, yet calpastatin levels were similar between treated and untreated mdx mice. These data demonstrate that pharmacological inhibition of calpain is not a promising intervention for the treatment of Duchenne muscular dystrophy due to the ability of skeletal muscle to counter calpain inhibitors by increasing multiple degradative pathways.

Rescue of dystrophic skeletal muscle by PGC-1α involves restored expression of dystrophin-associated protein complex components and satellite cell signaling

“…Novel therapies approved for human use have been slow in coming; however, numerous experimental approaches are present in the literature (1,5,38,39,(53)(54)(55). Among the most successful is replacing the missing dystrophin protein with utrophin, a dystrophin-related protein (23,46,60,63).…”

“…Muscle function (tetanic force, a series of eccentric contractions, and fatigue) was measured in the soleus at the Physiological Assessment Core of the Wellstone Muscular Dystrophy Cooperative Center at the University of Pennsylvania, according to standard techniques (3,4,36,39,(52)(53)(54)(55). Solei from different animals were used to measure eccentric injury and fatigue resistance, so that one measure would not interfere with the other.…”

“…In addition to a failure to maintain calcium homeostasis, metabolic dysregulation is also associated with the disease (17,32). The culmination of these cellular events is impaired muscle function (39,54,55), as contractile tissue is progressively replaced by adipose and fibrotic tissue (14). DMD is typically diagnosed in preschool-aged boys, after the child shows impaired motor function.…”

Proteomic assessment of the acute phase of dystrophin deficiency in mdx mice

“…We also detected increased expression of proteins involved in gene expression (Histone H4, Speckle-type POZ protein) and protein synthesis (Elongation factor 2). As activity of degradative pathways is known to be increased in dystrophic muscle (Selsby et al 2010;Spencer et al 1995) this is suggestive of increased protein turnover. Because both protein degradation and synthesis require ATP, this may represent an additional metabolic stress placed upon these muscles.…”

“…Finally, it could represent a significant pathological change, independent of fiber type specificity. For example, myosin is a calpain substrate (Lametsch et al 2004), and calpain activity is known to be increased in dystrophic skeletal muscle (Selsby et al 2010;Spencer et al 1995). A reduction in myosin heavy chain will undoubtedly contribute to reductions in muscle function (specific tension) seen in dystrophic muscle compared to control.…”