alpha-chymotrypsin and Myocardial-Ischemia

The heart is critically dependent on mitochondrial respiration for energy supply. Ischemia decreases oxygen availability, with catastrophic consequences for cellular energy systems. After a few minutes of ischemia, the mitochondrial respiratory chain halts, ATP levels drop and ion gradients across cell membranes collapse. Activation of cellular proteases and generation of reactive oxygen species by mitochondria during ischemia alter mitochondrial membrane permeability, causing mitochondrial swelling and fragmentation and eventually cell death. The mitochondria, therefore, are important targets of cardioprotection against ischemic injury. We have previously shown that ixazomib (IXA), a proteasome inhibitor used for treating multiple myeloma, effectively reduced the size of the infarct produced by global ischemia in isolated rat hearts and prevented degradation of the sarcoplasmic reticulum calcium release channel RyR2. The aim of this work was to further characterize the protective effect of IXA by determining its effect on mitochondrial morphology and function after ischemia. We also quantified the effect of IXA on levels of mitofusin-2, a protein involved in maintaining mitochondrial morphology and mitochondria-SR communication. We found that mitochondria were significantly preserved and functional parameters such as oxygen consumption, the ability to generate a membrane potential, and glutathione content were improved in mitochondria isolated from hearts perfused with IXA prior to ischemia. IXA also blocked the release of cytochrome c observed in ischemia and significantly preserved mitofusin-2 integrity. These beneficial effects resulted in a significant decrease in the left ventricular end diastolic pressure upon reperfusion and a smaller infarct in isolated hearts.

Topics: Animals; Boron Compounds; Chymotrypsin; Disease Models, Animal; Glutathione; Glycine; Heart; Humans; Membrane Potentials; Mitochondria; Myocardial Ischemia; Oxygen Consumption; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats

Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion.

Topics: Adenosine Triphosphate; Animals; Boron Compounds; Caspase 3; Cell Death; Chymotrypsin; Dose-Response Relationship, Drug; Enzyme Activation; Glycine; Heart; Hemodynamics; Male; Myocardial Ischemia; Myocardium; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; Ryanodine Receptor Calcium Release Channel

Arrhythmia-prone epicardial border zone (EBZ) tissues demonstrate decreased G protein-coupled receptor kinase-2 (GRK2) activity and increased sensitivity to isoproterenol 6-24 h after coronary artery ligation in the dog. We previously demonstrated that the ischemia-mediated decrease in GRK2 in cardiac ischemic tissue was largely blocked by proteasome blockade initiated 1 h before the onset of ischemia, and this was associated with significant cardioprotection against malignant ventricular tachyarrhythmias. For application to clinical circumstances, it is desirable to determine whether a clinical window exists following the onset of ischemia for such a protective effect. The treatment of six dogs with the selective proteasome inhibitor bortezomib 1 h after the surgical induction of left coronary artery ischemia provided 80% (EBZ) and 42% (infarct) protection (by immunoblot) against the loss of GRK2 at 24 h. There was no significant increase of heat shock protein 70(72) in the EBZ of bortezomib-treated animals compared with control. There was a striking absence of rapid (>300 beats/min) and very rapid (>360 beats/min) ventricular triplets that is highly predictive of sudden cardiac deaths (SCDs) during electrocardiogram monitoring of the first 24 h in the bortezomib-treated animals in contrast with nontreated infarcted animals. There were no SCDs in the 6 treated animals (0%) and five SCDs in the 14 control animals (36%). Assay of whole blood proteasome activity demonstrated the expected decrease over the 24-h observation period. These data support the concept that proteasome inhibition within a window of time following myocardial infarction may be of use in suppressing malignant tachyarrhythmias and SCD.

Topics: Animals; Blotting, Western; Boronic Acids; Bortezomib; Catecholamines; Chymotrypsin; Coronary Vessels; Death, Sudden, Cardiac; Dogs; Electrocardiography; Electrophysiology; G-Protein-Coupled Receptor Kinase 3; Heart Ventricles; HSP72 Heat-Shock Proteins; Ligation; Male; Myocardial Infarction; Myocardial Ischemia; Protease Inhibitors; Proteasome Inhibitors; Pyrazines; Tachycardia; Trypsin

The ubiquitin-proteasome system has been implicated in both cardiac physiology and pathophysiology. Research in this area has been hampered by the lack of a simple, reproducible method to assess 26S-proteasome peptidase activities. The current report demonstrates that one reason for lack of reproducibility is the myriad of ATP concentrations, many of them excessive, which have been used to stimulate peptidase activity. The chymotrypsin-like or caspase-like activities of 26S-proteasome in cardiac tissue isolates were determined using Suc-LLVY-AMC or Z-LLE-AMC, respectively, over a range of ATP concentrations up to 2 mmol/L. The optimal ATP concentration to assess both peptidase activities was found to be in the low micromolar range (from 6 to 100 micromol/L) depending on the cardiac tissue isolate protein (10 to 90 microg protein) contained in the reaction. Increasing ATP beyond the optimal range was inhibitory. In general, chymotrypsin-like and caspase-like activities could be stimulated 2- to 2.5-fold and 1.4- to 1.8-fold, respectively, over basal (ATP, 0 micromol/L), and could be effectively inhibited with lactacystin or Z-Pro-Nle-Asp-CHO, respectively. Based on these observations, an optimized method is presented for ex vivo determination of cardiac 26S-proteasome peptidase activities which was used to confirm inactivation of this complex by myocardial ischemia and reperfusion.

Topics: Adenosine Triphosphate; Animals; Caspases; Chymotrypsin; In Vitro Techniques; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley

Topics: Adolescent; Adult; Child; Chymotrypsin; Combined Modality Therapy; Female; Heterozygote; Homozygote; Humans; Hyperlipoproteinemia Type II; Male; Middle Aged; Myocardial Ischemia; Sorption Detoxification