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Calpain Activation Contributes to Endotoxin-Induced Diaphragmatic Dysfunction

¹ Division of Pulmonary, Critical Care, and Sleep Medicine, University of Kentucky, Lexington, Kentucky

Correspondence and requests for reprints should be addressed to Gerald S. Supinski, M.D., Department of Medicine, 740 South Limestone, K-528, University of Kentucky, Lexington, KY 40536-0284. E-mail: gsupi2{at}email.uky.edu

Calpain activation occurs in skeletal muscle in response to infection, but it is unknown if calpain inhibition improves muscle functional capacity. We hypothesized that infection induces diaphragm calpain activation, that calpain activation results in cleavage of important diaphragm cytoskeletal proteins, and that inhibition of calpain attenuates infection-induced diaphragm dysfunction. Mice (n = 4–6/group) were given: (1) saline (intraperitoneal); (2) endotoxin (12 mg/kg intraperitoneal); (3) calpain inhibitor peptide III (12 mg/kg intraperitoneal); and (4) endotoxin (12 mg/kg) plus calpain inhibitor peptide III (12 mg/kg). At 24 hours, diaphragms were removed and the following determined: (1) calpain activity by fluorogenic assay; (2) calpain I and II protein levels; (3) talin protein levels; and (4) the force–frequency relationship. Endotoxin significantly increased diaphragm calpain activity (P < 0.001), active calpain I protein (P < 0.001), active calpain II protein (P < 0.01), levels of a calpain-specific cleavage talin degradation product (P < 0.003), and reduced diaphragm force (P < 0.001). Calpain inhibitor III administration prevented endotoxin-induced increases in calpain activity, reduced talin degradation, and attenuated reductions in diaphragm force. Diaphragm-specific force at 150 Hz stimulation was significantly higher in control, endotoxin plus calpain inhibitor III, and calpain inhibitor III alone groups (23 ± 1, 20 ± 1 and 23 ± 1 N/cm², respectively) than in the endotoxin alone group (15 ± 1 N/cm²) (P < 0.01). This model of sepsis results in significant diaphragm calpain activation and calpain-dependent diaphragm cytoskeletal protein cleavage. Moreover, calpain inhibition attenuates endotoxin-induced diaphragm weakness, suggesting that such inhibitors may be a potential treatment to improve respiratory muscle function in infected patients.

Key Words: diaphragm • endotoxin • calpain • proteolysis

CLINICAL RELEVANCE Most critically ill patients have significant respiratory muscle weakness. This report identifies a potential therapeutic target that could be exploited to prevent weakness and improve respiratory function in this patient population.