Animals and ethics statement
All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals of the National Academy of Sciences (NIH publication No. 85-23, revised 1996). This study was carried out with the approval of the Animal Ethics Committee of Stellenbosch University.
Ex vivo perfusion protocol
Male Wistar rats weighing 180–220 g were used for this study. Rats were anesthetized (pentobarbitone, 100 mg/kg i.p.) and hearts rapidly excised and perfused in a modified Langendorff model as described before [21, 22]. Briefly, Krebs–Henseleit buffer containing (in mM) 11 glucose, 118 NaCl, 4.7 KCl, 1.2 MgSO4·7H2O, 2.5 CaCl2·2H2O, 1.2 KH2PO4, 25 NaHCO3 was equilibrated with 95% O2–5% CO2 (37°C, pH 7.4) at a constant pressure (100 cm). Buffer was not recirculated and the hearts were allowed to beat at their natural rate. During perfusion, a latex balloon attached to a pressure transducer (Stratham MLT 0380/D, ADInstruments Inc, Bella Vista, NSW, Australia) compatible with the PowerLab System ML410/W (ADInstruments Inc, Bella Vista, NSW, Australia) was inserted into the left ventricle via the mitral valve and inflated to produce a systolic pressure of 80–120 mmHg and a diastolic pressure of 4–12 mmHg. The temperature of the heart was maintained at 37°C by suspending it in a heated water jacket.
The protocol was divided into two parts, i.e. perfusions (a) with global ischemia and (b) regional ischemia, followed by reperfusion for cardiac functional assessments and infarct size determination, respectively. For each of these, hearts were randomly distributed into six experimental groups for perfusions: baseline control (11 mM glucose) ± either 5 μM lactacystin or 10 μM MG-132; and high glucose (33 mM glucose) ± either 5 μM lactacystin or 10 μM MG-132 (n = 8 rats were used for each of the experimental groups indicated). The respective UPS inhibitors (lactacystin, MG-132) were dissolved in the buffer and added for the first 20 min of reperfusion.
For our inhibition studies we aimed to achieve so-called “non-toxic proteasome inhibition’’, i.e. only partial attenuation of chymotrypsin-like activity as high and sustained doses can result in severe toxic effects with detrimental outcomes . Lactacystin is a natural compound that binds mainly to the β5 proteasomal subunit (responsible for chymotrypsin-like activity) of the 20S proteasome , leading to irreversible inhibition of chymotrypsin-like activity (reviewed in ). However, the trypsin- and caspase-like activities are blunted to a lesser extent in this instance . We also employed the reversible aldehyde inhibitor MG-132 that specifically binds to proteasomal subunit β5 of the 20S proteasome (reviewed in ). Proteasome inhibitor doses were selected to be in the range of what was employed in previously published studies [27, 28]. High glucose perfusions were used to simulate acute hyperglycemia and since ex vivo Langendorff perfusions are typically performed with 11 mM glucose at baseline, the 33 mM dose would be representative of a threefold elevation of glucose levels (above normal) within the clinical setting. Additional experiments were also performed in order to rule out the effects of osmotic pressure on heart function. Here rat hearts were perfused with 22 mM mannitol plus 11 mM glucose (total molarity = 33 mM) and subjected to ischemia–reperfusion as described above.
Ex-vivo global ischemia and reperfusion
The protocol included a 60 min stabilization period, 20 min of global ischemia, followed by 60 min of reperfusion. Contractile parameters assessed throughout the experiment included heart rate (HR), left ventricular developed pressure (LVDP), end diastolic pressure (EDP), rate-pressure product (RPP = HR × LVDP), and coronary flow. The percentage recovery for LVDP and RPP was also calculated where reperfusion data points were expressed as a percentage of pre-ischemic values. Coronary flow was measured by collection of the effluent at regular timed intervals. Both left and right ventricular tissues were immediately collected after the 60 min reperfusion period with tissues freeze-clamped in liquid nitrogen with pre-cooled Wollenberger tongs, whereafter it was stored at −80°C for further molecular and biochemical analyses.
Ex-vivo regional ischemia and reperfusion
To further strengthen our Langendorff functional data, we also evaluated the effects of lactacystin and MG-132 by infarct size determination. This was performed as described before [21, 22], and we employed regional ischemia with a reperfusion time of 2 h. Here a 3/0 silk suture was placed on the proximal portion of the left anterior descending coronary artery and passing the ends through a plastic tube. For induction of regional ischemia the ends were tightened by pressing the plastic tube against the surface of the heart (above the artery) for 20 min. The snare was released during the reperfusion period. The efficacy of ischemia was confirmed by regional cyanosis and a substantial decrease in coronary flow.
After completion of each regional ischemia–reperfusion experiment the snare was re-tightened and 2.5% Evans blue dye (in Krebs buffer) was perfused through the hearts for identification of the area at risk of ischemia. Hearts were subsequently removed from the Langendorff apparatus, blotted dry, suspended (using suture) within 50 ml plastic tubes and frozen at −20°C for 3 days. Frozen hearts were thereafter sliced into 2 mm transverse sections and incubated with 1% 2,3,5-triphenyl tetrazolium chloride in phosphate-buffered saline for 20 min at 37°C. Slices were then fixed in 10% formalin for 24 h at room temperature before being placed between glass plates for scanning. The infarct size and the area-at-risk were determined using Image J software (v1.46p, NIH, USA) and infarct size was expressed as a percentage of the area-at-risk.
Measurement of intracellular E3 ligases, oxidative stress, inflammation and autophagic markers
Protein isolation was performed as previously described . Briefly, collected heart tissues were homogenized with modified RIPA buffer, the supernatant centrifuged twice at 4,300g for 10 min at 4°C then stored at −80°C until further use. Protein expression was determined by SDS-PAGE as described previously by our laboratory [21, 29], and Western blotting performed with representative markers for: (a) E3 ligases; Muscle RING Finger 1 (MURF1; Abcam, Cambridge, MA, USA) and muscle atrophy F-box (MAFbx; Santa Cruz Biotechnologies, Santa Cruz, CA, USA); (b) oxidative stress: superoxide dismutase 1 and 2 (SOD1, SOD2; Santa Cruz Biotechnologies, Santa Cruz, CA, USA); (c) inflammation: tumor necrosis factor-alpha (TNF-α; Sigma-Aldrich, St. Louis, MO, USA), and inhibitor of nuclear factor kappa-B kinase subunit alpha (IκBα; Sigma-Aldrich, St. Louis, MO, USA); and (d) autophagy: microtubule‐associated protein 1 light chain 3 II (LC3-II; Cell Signaling, Danvers, MA, USA), and p62 (Cell Signaling, Danvers, MA, USA). Western blots were quantified by densitometric analysis and β-actin (Cell Signaling, Danvers, MA, USA) employed as a loading control as described before [21, 22].
Proteasome activity experiments
Heart tissues were cut into small slices and homogenized in 1 ml of Tris–HCl buffer (pH 7.4) using an IKA Ultra Turrax T25 homogenizer (IKA Labortechnik, Staufen, Germany) and incubated on ice for 10 min before centrifugation at 9,000g for 15 min to remove cell debris. The supernatant was employed for protein quantification using the Bradford assay as described previously .
The Proteasome-Glo™ 3-Substrate System (Promega, Madison, WI, USA) consists of three homogeneous bioluminescent assays that separately measure the three proteolytic activities associated with the proteasome, i.e. chymotrypsin-like (LLVY), trypsin-like (LSTR), and caspase-like (LLE) activities. The three assays differ in their ability to detect different protease activities based on their substrate components, i.e. the luminogenic substrates provided for the LLVY, LSTR, and LLE are Suc-LLVY-aminoluciferin, Z-LRR-aminoluciferin, and Z-nLPnLD-aminoluciferin, respectively. Each substrate is added to a buffer system optimized for proteasome and luciferase activities to make a Proteasome-Glo™ Reagent for a particular catalytic activity. The individual Proteasome-Glo™ Reagent is added to test samples in an “add-mix-measure” format, resulting in proteasome-induced cleavage of each particular substrate. Substrate cleavage generates a “glow-type” luminescent signal produced by the luciferase reaction that is proportional to proteasome activity.
Assays were performed with ~50 µg of protein lysate [in 25 mM Tris–HCl (pH 7.5)] together with the respective substrate—incubated together for 30 min at 37°C. Aminomethylcoumarin and β-naphthylamine luminescence was subsequently measured using a luminometer microplate reader (BMG Labtech, Ortenberg, Germany) and data were normalized to protein concentrations.
Data are presented as mean ± standard error of mean (SEM). Statistical analysis was performed by the Mann–Whitney t-test, or one-way analysis of variance (ANOVA) followed by the Tukey–Kramer post hoc test (GraphPad Prism v5, San Diego, CA, USA). Values were considered significant when p < 0.05.