Calpain Activation Contributes to Endotoxin-Induced Diaphragmatic Dysfunction

Abstract

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 5 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 6 1, 20 6 1 and 23 6 1 N/cm 2 , respectively) than in the endotoxin alone group (15 6 1 N/cm 2 ) (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.

The Sick and the Weak: Neuropathies/Myopathies in the Critically Ill

“…Greater calpain activity leads to Z-band disintegration and myofibrillar protein breakdown (202,776), thereby depressing myofibrillar force (674). Calpain inhibitors lessen sepsis effects on muscle, reducing proteolysis (191,767) and preserving contractile function (675,685). The mechanism by which sepsis increases calpain activity is less clear.…”

Molecular mechanisms of intensive care unit-acquired weakness

“…Activation of calpains also reduces Akt phosphorylation and may therefore contribute to decreased protein synthesis [87]. Increased calpain activity decreased contractile function in septic models of muscle wasting [88,89]; thus it is possible that activation of calpains contributes to the early loss in force generation, as well as contributing to proteolysis.…”

“…For example, oxidative stress increases ubiquitination of cellular proteins and also activity of the UP itself [122,123]. Similarly, both calpains and caspases can be induced by free radicals [88,124,125] and free radicals have been shown to cause sarcolemmal damage, which can be reduced by inhibition of nitric oxide synthase [101]. Finally, it has also been suggested that free radicals may interact directly with the contractile apparatus of the myofilament and by modifying the protein structure, reduce contractile potential [126].…”

“…Free radical production is increased from each of these sources in sepsis [127][128][129]. In animal models, reversal of some of the pathological effects of sepsis can be achieved with free radical scavengers and inhibitors of free radical production [88,130,131]. Although the potential of antioxidant therapy has been demonstrated in some causes of muscle atrophy, as yet there is no evidence for this in ICUAW [132].…”

Calpain Inhibition Preserves Talin and Attenuates Right Heart Failure in Acute Pulmonary Hypertension

“…Although the overall difference in talin abundance was modest, relatively small changes in major structural proteins can lead to large changes in function. For example, Supinski and Callahan found that a 30% difference in talin abundance was associated with a 30% difference in contractile function of rat diaphragm muscle after experimental endotoxemia (33). a-actinin, a major adhesion protein found predominantly at the sarcomeric z-disk that is sensitive to calpain, was similarly altered, as assessed by immunohistology, but not by Western blotting, consistent with reports by other investigators that calpain can induce major structural changes in some proteins without degrading them (34).…”