title: Sepsis stimulates calpain activity in skeletal muscle by decreasing calpastatin activity but does not activate caspase-3.
abstract: We examined the influence of sepsis on the expression and activity of the calpain and caspase systems in skeletal muscle. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham operated. Calpain activity was determined by measuring the calcium-dependent hydrolysis of casein and by casein zymography. The activity of the endogenous calpain inhibitor calpastatin was measured by determining the inhibitory effect on calpain activity in muscle extracts. Protein levels of mu- and m-calpain and calpastatin were determined by Western blotting, and calpastatin mRNA was measured by real-time PCR. Caspase-3 activity was determined by measuring the hydrolysis of the fluorogenic caspase-3 substrate Ac-DEVD-AMC and by determining protein and mRNA expression for caspase-3 by Western blotting and real-time PCR, respectively. In addition, the role of calpains and caspase-3 in sepsis-induced muscle protein breakdown was determined by measuring protein breakdown rates in the presence of specific inhibitors. Sepsis resulted in increased muscle calpain activity caused by reduced calpastatin activity. In contrast, caspase-3 activity, mRNA levels, and activated caspase-3 29-kDa fragment were not altered in muscle from septic rats. Sepsis-induced muscle proteolysis was blocked by the calpain inhibitor calpeptin but was not influenced by the caspase-3 inhibitor Ac-DEVD-CHO. The results suggest that sepsis-induced muscle wasting is associated with increased calpain activity, secondary to reduced calpastatin activity, and that caspase-3 activity is not involved in the catabolic response to sepsis.
authors.affiliation: Dept. of Surgery, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Wei
authors.forename: Moin U
authors.initials: MU
authors.lastname: Fareed
authors.forename: Amy
authors.initials: A
authors.lastname: Evenson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Menconi
authors.forename: Hongmei
authors.initials: H
authors.lastname: Yang
authors.forename: Victoria
authors.initials: V
authors.lastname: Petkova
authors.forename: Per-Olof
authors.initials: PO
authors.lastname: Hasselgren
mesh_terms: D000818:Animals; D001424:Bacterial Infections / Q000201:enzymology / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D008297:Male; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism; D009124:Muscle Proteins / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000201:enzymology / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D009124:Muscle Proteins; D009842:Oligopeptides; D012333:RNA, Messenger; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C071482:calpeptin; C033668:calpastatin; D002154:Calpain; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases