Ischemia time impacts on respiratory chain functions and Ca2+-handling of cardiac subsarcolemmal mitochondria subjected to ischemia reperfusion injury.

BACKGROUND: Mitochondrial impairment can result from myocardial ischemia reperfusion injury (IR). Despite cardioplegic arrest, IR-associated cardiodepression is a major problem in heart surgery. We determined the effect of increasing ischemia time on the respiratory chain (RC) function, the inner membrane polarization and Ca2+ homeostasis of rat cardiac subsarcolemmal mitochondria (SSM). METHODS: Wistar rat hearts were divided into 4 groups of stop-flow induced warm global IR using a pressure-controlled Langendorff system: 0, 15, 30 and 40 min of ischemia with 30 min of reperfusion, respectively. Myocardial contractility was determined from left ventricular pressure records (dP/dt, dPmax) with an intraventricular balloon. Following reperfusion, SSM were isolated and analyzed regarding electron transport chain (ETC) coupling by polarography (Clark-Type electrode), membrane polarization (JC1 fluorescence) and Ca2+-handling in terms of Ca2+-induced swelling and Ca2+-uptake/release (Calcium Green-5 N® fluorescence). RESULTS: LV contractility and systolic pressure during reperfusion were impaired by increasing ischemic times. Ischemia reduced ETC oxygen consumption in IR40/30 compared to IR0/30 at complex I-V (8.1 ± 1.2 vs. 18.2 ± 2.0 nmol/min) and II-IV/V (16.4 ± 2.6/14.8 ± 2.3 vs. 2.3 ± 0.6 nmol/min) in state 3 respiration (p < 0.01). Relative membrane potential revealed a distinct hyperpolarization in IR30/30 and IR40/30 (171.5 ± 17.4% and 170.9 ± 13.5%) compared to IR0/30 (p < 0.01), wearing off swiftly after CCCP-induced uncoupling. Excess mitochondrial permeability transition pore (mPTP)-gated Ca2+-induced swelling was recorded in all groups and was most pronounced in IR40/30. Pyruvate addition for mPTP blocking strongly reduced SSM swelling in IR40/30 (relative AUC, ± pyruvate; IR0/30: 1.00 vs. 0.61, IR15/30: 1.68 vs. 1.00, IR30/30: 1.42 vs. 0.75, IR40/30: 1.97 vs. 0.85; p < 0.01). Ca2+-uptake remained unaffected by previous IR. Though Ca2+-release was delayed for ≥30 min of ischemia (p < 0.01), Ca2+ retention was highest in IR15/30 (RFU; IR0/30: 6.3 ± 3.6, IR 15/30 42.9 ± 5.0, IR30/30 15.9 ± 3.8, IR40/30 11.5 ± 6.6; p ≤ 0.01 for IR15/30 against all other groups). CONCLUSIONS: Ischemia prolongation in IR injury gradually impaired SSM in terms of respiratory chain function and Ca2+-homeostasis. Membrane hyperpolarization appears to be responsible for impaired Ca2+-cycling and ETC function. Ischemia time should be considered an important factor influencing IR experimental data on subsarcolemmal mitochondria. Periods of warm global ischemia should be minimized during cardiac surgery to avoid excessive damage to SSMs.

Chemically monoubiquitinated PEX5 binds to the components of the peroxisomal docking and export machinery.

Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. "Click" chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ΔPEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interactions with the docking proteins were independent of the PEX5 ubiquitination status whereas the interactions with the REM components were increased when PEX5 is ubiquitinated.

Cardiac Surgery is Safe in Female Patients with a History of Breast Cancer.

PURPOSE: In cardiac surgery candidates, a concomitant history of breast cancer suggests adverse outcomes. The possibility of internal mammary artery (IMA) utilization and its patency rate is frequently discussed. Secondary, blood loss and wound related infections might be important issues. However, publications focusing on these issues are limited. METHODS: We analyzed 32 patients with previously treated breast cancer undergoing cardiac bypass (CABG) and combined CABG surgery matched to 99 control subjects in a retrospective cohort study. Patients were analyzed regarding IMA utilization, blood loss and substitution and frequent perioperative complications as well as long-term mortality. RESULTS: No significant differences between groups were observed regarding duration of surgery, IMA-utilization, incidence of infections and postoperative complications or mortality. A pronounced decline of hemoglobin/hematocrit was evident within the first 6 postoperative hours (3.3 ± 1.8 vs. 2.5 ± 1.8 mg/dl; p = 0.03) in breast cancer patients not related to an increased drainage loss but associated with an increase of international normalized ratio (INR) (0.39 ± 0.16 vs. 0.29 ± 0.24; p <0.01). CONCLUSION: In breast cancer patients, CABG and combined CABG procedures can safely be performed with comparable short- and long-term results.

Impact of levosimendan and ischaemia-reperfusion injury on myocardial subsarcolemmal mitochondrial respiratory chain, mitochondrial membrane potential, Ca2+ cycling and ATP synthesis.

OBJECTIVES: Levosimendan (LS) is increasingly used in case of myocardial failure after cardiac surgery. The impact of LS on myocardial mitochondrial functions, such as respiratory chain function (RCF), mitochondrial membrane potential (ΔΨm), Ca(2+) handling, mitochondrial permeability transition pore (mPTP) opening and ATP during ongoing ischaemia/reperfusion (IR) injury, is not well understood. Depending on LS, I/R injury or the combination of both, we analysed myocardial functions in a retrograde Langendorff-model followed by the analysis of subsarcolemmal mitochondrial (SSM) functions. METHODS: Rat hearts were divided into four study groups; two were subjected to 30 min of perfusion without (control) or with the application of 1.4 µmol/20 min LS (Levo). Experiments were repeated with hearts being subjected to 40 min of normothermic stop-flow ischaemia and 30 min of reperfusion without (IR) or with LS application (Levo-IR). Systolic left ventricular pressure (LVPsys), left ventricular contractility (LVdp/dtmax) and coronary flow were determined. SSM were analysed regarding RCF, ΔΨm, ATP, and Ca(2+) retention capacity (CRC), Ca(2+)-induced swelling and Ca(2+) fluxes after (re)perfusion. RESULTS: I/R injury suppressed LVdp/dtmax (1381 ± 927 vs 2464 ± 913 mmHg/s; P = 0.01 at 30 min (re-)perfusion time). IR revealed complex I-V state3 (19.1 ± 7.4 vs 27.6 ± 11.0 nmolO2/min; P < 0.044) and II-V state3 (20.6 ± 6.8 vs 37.3 ± 9.10 molO2/min; P < 0.0001) suppression and Levo limited I-V (14.8 ± 11.1 vs 27.6 ± 11.0 nmolO2/min; P < 0.001) and II-V (24.1 ± 6.4 vs 37.3 ± 9.10 molO2/min; P < 0.0001) function. After energizing, ΔΨm hypopolarization was observed in Levo (0.76 ± 0.04 vs 0.84 ± 0.04; P = 0.02), IR (0.75 ± 0.06 vs 0.84 ± 0.04; P = 0.007) and Levo-IR (0.75 ± 0.06 vs 0.06 ± 0.04; P = 0.01). IR (AUC: 626 vs 292; P = 0.023) and Levo-IR (AUC: 683 vs 292, P = 0.003) increased Ca(2+)-induced mPTP-opening susceptibility. CRC declined in IR (6.4 ± 2.1 vs 10.5 ± 2.6; P = 0.04) or Levo (6.5 ± 2.0 vs 10.5 ± 2.6; P = 0.023). Ca(2+) uptake was delayed in IR and Levo-IR without LS impact (P < 0.0001). Ca(2+) liberation was increased in Levo-IR. ATP synthesis was reduced in Levo (0.49 ± 0.14 vs 0.74 ± 0.14; P = 0.002) and Levo-I/R (0.34 ± 0.18 vs 0.74 ± 0.14; P < 0.002). CONCLUSION: LS limited RCF at complex IV and V with ΔΨm hypopolarization suggesting a specific [Formula: see text]-dependent pathway. Ca(2+) redistribution from SSM by LS during I/R injury possibly prevents from Ca(2+) overload due to mPTP flickering. LS-induced mPTP flickering did not promote permanent Ca(2+)-induced mPTP opening. LS-dependent inhibition of ATP generation presumably resulted from complex IV and V limitations and lowered ΔΨm. However, a resulting impact of limited ATP synthesis on myocardial recovery remains arguable.

A stable chemical SUMO1-Ubc9 conjugate specifically binds as a thioester mimic to the RanBP2-E3 ligase complex.

Ubiquitin and ubiquitin-like (Ubl) modifiers such as SUMO are conjugated to substrate proteins by E1, E2, and E3 enzymes. In the presence of an E3 ligase, the E2∼Ubl thioester intermediate becomes highly activated and is prone to chemical decomposition, thus making biochemical and structural studies difficult. Here we explored a stable chemical conjugate of the E2 enzyme from the SUMO pathway, Ubc9, with its modifier SUMO1 as a structural analogue of the Ubc9∼SUMO1 thioester intermediate, by introducing a triazole linkage by biorthogonal click chemistry. The chemical conjugate proved stable against proteolytic cleavage, in contrast to a Ubc9-SUMO1 isopeptide analogue obtained by auto-SUMOylation. Triazole-linked Ubc9-SUMO1 bound specifically to the preassembled E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9, thus suggesting that it is a suitable thioester mimic. We anticipate interesting prospects for its use as a research tool to study protein complexes involving E2 and E3 enzymes.

Covalent inhibition of SUMO and ubiquitin-specific cysteine proteases by an in situ thiol-alkyne addition.

Posttranslational modification of proteins with ubiquitin and ubiquitin-like modifiers such as SUMO can be reverted by specific proteases, also referred to as deubiquitinases and isopeptidases, most of which are cysteine-dependent. We have found that the replacement of the conserved C-terminal glycine with propargylamine converts SUMO and ubiquitin to highly efficient covalent inhibitors of their cognate cysteine proteases. Attack of the catalytic cysteine onto the terminal alkyne results in the formation of a vinyl sulfide linkage. Although this reaction is reminiscent of the inhibitory mechanism of the isosteric nitrile inhibitors it was unexpected due to the low electrophilicity of the alkyne group. We show that a precise location of the functional group in the active site of the protease is crucial for the reaction, which was not inhibited by the presence of a radical scavenger. Furthermore, a mutational study of key catalytic residues in the SUMO-protease Senp1, that is H533A and D550A of the catalytic triad and Q597A as part of the oxyanion hole, revealed that these residues are not required for the observed covalent adduct formation. We therefore propose that the reaction is an in situ thiol-alkyne addition. Due to the high chemical inertness of the alkyne moiety the respective protease inhibitors should be well-suited for cellular and therapeutic applications. In keeping with this idea, selective labeling with propargylated SUMO and Ub probes was observed in lysates of cell lines expressing the cognate proteases after transient transfection.

Cardiac surgery in cases of myeloproliferative neoplasm: risk factor for stroke.

OBJECTIVES: a history of myeloproliferative neoplasms is considered to increase the risks in cardiac surgery. In patients with myeloproliferative neoplasms, increased rates of perioperative infections and thromboembolic complications are suspected, but studies analyzing the impact of myeloproliferative neoplasms on results after cardiac surgery are lacking. METHODS: 13 patients with the diagnosis of myeloproliferative neoplasm underwent cardiac surgery. These patients were matched to 36 controls. Matching criteria consisted of sex, age, diagnosis, and comorbidities. Patients were analyzed regarding laboratory parameters, blood transfusion demands, morbidity, and mortality. RESULTS: compared to controls, patients with myeloproliferative neoplasms demonstrated a significantly lower body-mass index (p<0.01), creatinine (p=0.024), prothrombin time (p=0.001), and urea level (p=0.012). The perioperative leukocyte response (p=0.03) was ameliorated, and platelet counts (p<0.02) increased. Patients with myeloproliferative neoplasms had a reduced need for erythrocyte concentrates (54% vs. 86%, p=0.047) but increased need for plasma and thrombocytes (15% vs. 0%, p=0.07). Patients with myeloproliferative neoplasms had a significantly increased incidence of thromboembolic events compared to controls (31% vs. 3%, p=0.014). Hospital mortality remained at zero, but mid-term survival was lower in patients with myeloproliferative neoplasms (p=0.078). CONCLUSIONS: myeloproliferative neoplasm as a concomitant diagnosis increases the risk of thromboembolic complications during cardiac surgery. Plasma and platelet substitutions have to be administered, although strokes were not associated with hemostatic treatment.

Glycine preconditioning to ameliorate pulmonary ischemia reperfusion injury in rats.

This study examines the impact of glycine (Gly) preconditioning on ischemia reperfusion (IR)-induced pulmonary mitochondrial injury to research the previously, in pig lungs, demonstrated Gly-dependent amelioration of pulmonary IR injury. IR injury was induced in rat lungs by 30 min pulmonary hilum clamping followed by 60 min reperfusion time. Rats were subjected to controls, shams and two study groups (IR30/60, Gly-IR30/60) receiving 37.5 mg Gly i.v. or not before IR induction. The wet/dry-weight ratio, mitochondria viability (MV), membrane integrity (MI), respiratory chain complex (RCC) activities, mitochondrial membrane potential (ΔΨm) and cytochrome C (Cyt C) content were analysed. In IR30/60, RCC and MV were impaired; Cyt C loss and MI combined with matrix metalloproteinase-9 (MMP-9) activation and ΔΨm alteration were observed when compared with controls. In Gly-IR30/60, complex II function and mitochondrial viability were protected during IR, and MMP-9 activation combined with tissue-water content accumulation and ΔΨm alteration were ameliorated. Cyt C loss, mitochondrial membranes damage, tissue GSH oxidation or neutrophil sequestration was not extenuated in Gly-IR30/60. Gly ameliorates IR-associated mitochondrial dysfunction and decay of viability and normalizes ΔΨm but does not protect from Cyt C liberation and mitochondrial membrane damage. Our data suggest that the previously described effect of Gly preconditioning results at least partially from mitochondrial protection. A dose-finding study is necessary to improve results of Gly preconditioning.

Glutathione preconditioning ameliorates mitochondria dysfunction during warm pulmonary ischemia-reperfusion injury.

OBJECTIVES: Reduced glutathione (GSH) has been shown to improve pulmonary graft preservation. Mitochondrial dysfunction is regarded to be the motor of ischemia-reperfusion injury (IR) in solid organs. We have shown previously that IR induces pulmonary mitochondrial damage. This study elucidates the impact of GSH preconditioning on the integrity and function of pulmonary mitochondria in the setting of warm pulmonary IR. METHODS: Wistar rats were subjected to control, sham, and to two-study-group conditions (IR30/60 and GSH-IR30/60) receiving IR with or without GSH preconditioning. Rats were anesthetized and received mechanical ventilation. Pulmonary in situ clamping followed by reperfusion generated IR. Mitochondria were isolated from pulmonary tissue. Respiratory chain complexes activities (I-IV) were analyzed by polarography. Mitochondrial viability (Ca2+-induced swelling) and membrane integrity (citrate synthase assay) were determined. Subcellular-fractional cytochrome C-content (Cyt C) was quantified by enzyme-linked immunosorbent assay (ELISA). Mitochondrial membrane potential (ΔΨm) was analyzed by fluorescence-activated cell sorting (FACS) after energizing and uncoupling. Inflammatory activation was determined by myeloperoxidase activity (MPO), matrix-metalloproteinase 9 (MMP-9) activity by gel zymography. RESULTS: Pulmonary IR significantly reduced mitochondrial viability in combination with ΔΨm hyper-polarization. GSH preconditioning improved mitochondrial viability and normalized ΔΨm. Cyt C was reduced after IR; GSH protected from Cyt C liberation. Respiratory chain complex activities (I, II, III) declined during IR; GSH protected complex II function. GSH also protected from MMP-9 and neutrophil sequestration (P>.05). CONCLUSIONS: GSH preconditioning is effective to prevent mitochondrial death and improves complex II function during IR, but not mitochondrial membrane stability. GSH-mediated amelioration of ΔΨm hyper-polarization appears to be the key factor of mitochondrial protection.

Click synthesis of ubiquitin dimer analogs to interrogate linkage-specific UBA domain binding.

A new route to the synthesis of triazole-linked ubiquitin dimers (diUbs) as structural analogs of the seven diUbs is reported. Binding studies with the Lys48-specific UBA domain of the Mud1 protein suggest that they represent functionally suitable surrogates of their native counterparts linked by an isopeptide bond.

Expanded click conjugation of recombinant proteins with ubiquitin-like modifiers reveals altered substrate preference of SUMO2-modified Ubc9.

Wrestling with SUMO: the chemical conjugation of proteins with small ubiquitin-like modifiers (SUMO) can be achieved by a copper(I)-catalyzed cycloaddition and unnatural amino acid mutagenesis. This approach overcomes previous restrictions related to the primary sequence of proteins and coupling conditions. Moreover, biochemical data suggests that this triazole linkage presents the modifier in a proper distance and orientation relative to the target protein.

Ischemia-reperfusion injury-induced pulmonary mitochondrial damage.

BACKGROUND: Mitochondrial dysfunction is a key factor in solid organ ischemia-reperfusion (IR) injury. Impaired mitochondrial integrity predisposes to cellular energy depletion, free radical generation, and cell death. This study analyzed mitochondrial damage induced by warm pulmonary IR. METHODS: Anesthetized Wistar rats received mechanical ventilation. Pulmonary clamping was followed by reperfusion to generate IR injury. Rats were subjected to control, sham, and to 2 study group conditions: 30 minutes of ischemia without reperfusion (IR30/0), or ischemia followed by 60 minutes of reperfusion (IR30/60). Pulmonary edema was quantified by wet/dry-weight ratio. Polarography determined activities of respiratory chain complexes. Mitochondrial viability was detected by using Ca(2+)-induced swelling, and integrity by citrate synthase assay. Enzyme-linked immunosorbent assay determined cytochrome C content. Mitochondrial membrane potential (ΔΨm) stability was analyzed by flow cytometry using JC1, inflammation by myeloperoxidase (MPO) activity, and matrix-metalloproteinase-9 (MMP-9) activity by gel zymography, respectively. RESULTS: In IR30/60 rats, tissue water content was elevated from 80.6 % (sham) to 86.9%. After ischemia, ΔΨm showed hyperpolarization and rapid decline after uncoupling compared with controls. IR, but not ischemia alone, impaired respiratory chain function complexes I, II and III (p < 0.05). Mitochondrial viability (p < 0.001) and integrity (p < 0.01) was impaired after ischemia and IR, followed by mitochondrial cytochrome C loss (p < 0.05). Increased activation of MPO (p < 0.01) and MMP-9 (p < 0.001) was induced by reperfusion after ischemia. CONCLUSIONS: Ischemia-related ΔΨm hyper-polarization induces reperfusion-associated mitochondrial respiratory chain dysfunction in parallel with tissue inflammation and degradation. Controlling ΔΨm during ischemia might reduce IR injury.

Surfactant protein D increases phagocytosis and aggregation of pollen-allergen starch granules.

Recent studies have shown that surfactant components, in particular the collectins surfactant protein (SP)-A and -D, modulate the phagocytosis of various pathogens by alveolar macrophages. This interaction might be important not only for the elimination of pathogens but also for the elimination of inhaled allergens and might explain anti-inflammatory effects of SP-A and SP-D in allergic airway inflammation. We investigated the effect of surfactant components on the phagocytosis of allergen-containing pollen starch granules (PSG) by alveolar macrophages. PSG were isolated from Dactylis glomerata or Phleum pratense, two common grass pollen allergens, and incubated with either rat or human alveolar macrophages in the presence of recombinant human SP-A, SP-A purified from patients suffering from alveolar proteinosis, a recombinant fragment of human SP-D, dodecameric recombinant rat SP-D, or the commercially available surfactant preparations Curosurf and Alveofact. Dodecameric rat recombinant SP-D enhanced binding and phagocytosis of the PSG by alveolar macrophages, whereas the recombinant fragment of human SP-D, SP-A, or the surfactant lipid preparations had no effect. In addition, recombinant rat SP-D bound to the surface of the PSG and induced aggregation. Binding, aggregation, and enhancement of phagocytosis by recombinant rat SP-D was completely blocked by EDTA and inhibited by d-maltose and to a lesser extent by d-galactose, indicating the involvement of the carbohydrate recognition domain of SP-D in these functions. The modulation of allergen phagocytosis by SP-D might play an important role in allergen clearance from the lung and thereby modulate the allergic inflammation of asthma.