Role of calpain in skeletal-muscle protein degradation

Although protein degradation is enhanced in muscle-wasting conditions and limits the rate of muscle growth in domestic animals, the proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. The goals of this study were to evaluate the roles of the calpains (calcium-activated cysteine proteases) in mediating muscle protein degradation and the extent to which these proteases participate in protein turnover in muscle. Two strategies to regulate intracellular calpain activities were developed: overexpression of dominant-negative m-calpain and overexpression of calpastatin inhibitory domain. To express these constructs, L8 myoblast cell lines were transfected with LacSwitch plasmids, which allowed for isopropyl β-d-thiogalactoside-dependent expression of the gene of interest. Inhibition of calpain stabilized fodrin, a well characterized calpain substrate. Under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain reduced protein degradation by 30%, whereas calpastatin inhibitory domain expression reduced degradation by 63%. Inhibition of calpain also stabilized nebulin. These observations indicate that calpains play key roles in the disassembly of sarcomeric proteins. Inhibition of calpain activity may have therapeutic value in treatment of muscle-wasting conditions and may enhance muscle growth in domestic animals.

Functional cleavage of the common cytokine receptor γ chain (γc) by calpain

The small subunit of calpain, a calcium-dependent cysteine protease, was found to interact with the cytoplasmic domain of the common cytokine receptor γ chain (γc) in a yeast two-hybrid interaction trap assay. This interaction was functional as demonstrated by the ability of calpain to cleave in vitro-translated wild-type γc, but not γc containing a mutation in the PEST (proline, glutamate, serine, and threonine) sequence in its cytoplasmic domain, as well as by the ability of endogenous calpain to mediate cleavage of γc in a calcium-dependent fashion. In T cell receptor-stimulated murine thymocytes, calpain inhibitors decreased cleavage of γc. Moreover, in single positive CD4+ thymocytes, not only did a calpain inhibitor augment CD3-induced proliferation, but antibodies to γc blocked this effect. Finally, treatment of cells with ionomycin could inhibit interleukin 2-induced STAT protein activation, but this inhibition could be reversed by calpain inhibitors. Together, these data suggest that calpain-mediated cleavage of γc represents a mechanism by which γc-dependent signaling can be controlled.

Calpain inhibitor AK295 attenuates motor and cognitive deficits following experimental brain injury in the rat.

Marked increases in intracellular calcium may play a role in mediating cellular dysfunction and death following central nervous system trauma, in part through the activation of the calcium-dependent neutral protease calpain. In this study, we evaluated the effect of the calpain inhibitor AK295 [Z-Leu-aminobutyric acid-CONH(CH2)3-morpholine] on cognitive and motor deficits following lateral fluid percussion brain injury in rats. Before injury, male Sprague-Dawley rats (350-425 g) were trained to perform a beam-walking task and to learn a cognitive test using a Morris water maze paradigm. Animals were subjected to fluid percussion injury (2.2-2.4 atm; 1 atm = 101.3 kPa) and, beginning at 15 min postinjury, received a continuous intraarterial infusion of AK295 (120-140 mg/kg, n = 15) or vehicle (n= 16) for 48 hr. Sham (uninjured) animals received either drug (n = 5) or vehicle (n = 10). Animals were evaluated for neurobehavioral motor function at 48 hr and 7 days postinjury and were tested in the Morris water maze to evaluate memory retention at 7 days postinjury. At 48 hr, both vehicle- and AK295-treated injured animals showed significant neuromotor deficits (P< 0.005). At 7 days, injured animals that received vehicle continued to exhibit significant motor dysfunction (P< 0.01). However, brain-injured, AK295-treated animals showed markedly improved motor scores (P<0.02), which were not significantly different from sham (uninjured) animals. Vehicle-treated, injured animals demonstrated a profound cognitive deficit (P< 0.001), which was significantly attenuated by AK295 treatment (P< 0.05). To our knowledge, this study is the first to use a calpain inhibitor following brain trauma and suggests that calpain plays a role in the posttraumatic events underlying memory and neuromotor dysfunction.