Epoxomicin

To prevent MV-induced proteasome activation in the diaphragm, we administered the highly selective proteasome inhibitor, epoxomicin (0.55 μg/kg body weight) (Boston Biochem, Boston, MA). This dose was chosen based on previous reports demonstrating the effectiveness of this dose 14 and the efficacy of this dose was confirmed in our preliminary experiments. The inhibitor was dissolved in 60% dimethyl sulfoxide (DMSO) and given intravenously as a bolus immediately prior to the initiation of MV. Importantly, preliminary work demonstrated that the use of DMSO as the vehicle had no additional effects on diaphragm contractile dysfunction following 12 hours of MV (Table 1).

Tissue slices (6 pooled slices/sample) were lysed in ice-chilled buffer containing 20mM Tris-HCl pH7.5, 1mM MgCl₂, 1mM EDTA and 1mM DTT. Lysates were cleared by centrifugation and protein concentration was measured with the Bio-Rad protein assay. Chymotryptic, tryptic and caspase-like proteasomal activities were determined in lysates by cleavage of amino-4-methylcoumarin (AMC) from fluorogenic peptide substrates Suc-LLVY-7-AMC, Ac-RLR-AMC and Z-LLE-AMC (all Boston Biochem, Cambridge, MA), respectively. 20 μg total protein was pre-incubated at 37°C for 2min before addition of 10μM substrate peptides. Thereafter, AMC fluorescence was continuously measured for lmin at 37°C by using an Ex/Em 380/460nm filter set on a F-2500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). AMC fluorescence generated over time was considered directly proportional to proteasomal catalytic activity in samples. Verifying functionality of the assay, addition of 10μM proteasome inhibitor lactacystin (Boston Biochem) to the reaction completely inhibited generation of AMC fluorescence. Changes in proteasomal activities in slices after ischemia, after induction of ROS production with H₂O₂ or X/XO, or after treatment with the proteasome inhibitor epoxomicin (Boston Biochem) were calculated relative to control conditions.