leupeptins and Myocardial-Infarction

Clusterin (CLU) is induced in many organs after tissue injury or remodeling. Recently, we show that CLU levels are increased in plasma and left ventricle (LV) after MI, however, the mechanisms involved are not yet elucidated. On the other hand, it has been shown that the activity of the protein degradation systems (PDS) is affected after MI with a decrease in ubiquitin proteasome system (UPS) and an increase in macroautophagy. The aim of this study was to decipher if the increased CLU levels after MI are in part due to the alteration of PDS activity. Rat neonate cardiomyocytes (NCM) were treated with different modulators of UPS and macroautophagy in order to decipher their role in CLU expression, secretion, and degradation. We observed that inhibition of UPS activity in NCM increased CLU mRNA levels, its intracellular protein levels (p-CLU and m-CLU) and its secreted form (s-CLU). Macroautophagy was also induced after MG132 treatment but is not active. The inhibition of macroautophagy induction in MG132-treated NCM increased CLU mRNA and m-CLU levels, but not s-CLU compared to NCM only treated by MG132. We also demonstrate that CLU can be degraded in NCM through proteasome and lysosome by a macroautophagy independent pathway. In another hand, CLU silencing in NCM has no effect either on macroautophagy or apoptosis induced by MG132. However, the overexpression of CLU secreted isoform in H9c2 cells, but not in NCM decreased apoptosis after MG132 treatment. Finally, we observed that increased CLU levels in hypertrophied NCM and in failing human hearts are associated with proteasome inhibition and macroautophagy alteration. All these data suggest that increased CLU expression and secretion after MI is, in part, due to a defect of UPS and macroautophagy activities in the heart and may have a protective effect by decreasing apoptosis induced by proteasome inhibition.

Topics: Animals; Apoptosis; Autophagy; Biopsy; Clusterin; Gene Silencing; Heart Failure; Humans; Hypertrophy; Leupeptins; Lysosomes; Myocardial Infarction; Myocardium; Myocytes, Cardiac; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis; Rats; Ubiquitinated Proteins; Ubiquitination

Topics: Animals; Glycogen Synthase Kinase 3; GTP Phosphohydrolases; Humans; Indoles; Leptin; Leupeptins; Male; Maleimides; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Metalloendopeptidases; Mice; Mitochondrial Proteins; Myocardial Infarction; Proteolysis; Signal Transduction; Ubiquitination

To test the hypothesis that cellular proteinases contribute to ischemic myocellular death, measurements were made of tyrosine release (an index of overall proteolysis) from incubated slices of nonischemic and ischemic myocardium obtained at various times after coronary artery occlusion in rats. Proteolysis failed to increase in ischemic myocardium throughout the first 24 hours of occlusion, when irreversible damage develops, indicating that cellular proteinases do not undergo generalized activation in this phase. These data represent the first assessment of myocardial proteolysis throughout the development of ischemic death, and suggest that cellular proteinases do not play a causal role in this process. However, the possibility remains that ischemia selectively accelerates the breakdown of vital proteins, a phenomenon that may not be detected by measuring overall proteolysis. To determine whether future studies on the effects of proteolytic inhibition on infarct size are feasible, the ability of the proteinase inhibitors antipain, leupeptin, pepstatin and chymostatin, given in vivo, to interfere with proteolysis in ischemic myocardium was also evaluated. Leupeptin (10 or 40 mg/kg) inhibited proteolysis in a dose-related fashion (-49 and -72%, respectively, p less than 0.001). Antipain (20 mg/kg) decreased protein breakdown by 60% (p less than 0.001). The combination of antipain (20 mg/kg), leupeptin (40 mg/kg) and pepstatin (5 mg/kg) suppressed proteolysis almost completely at both 15 minutes (-88%, p less than 0.001) and at 6 hours (-72%, p less than 0.05) of ischemia, that is, throughout the development of irreversible injury. These results demonstrate that whatever proteolysis is occurring during acute myocardial infarction is largely mediated by cathepsins A, B, D, L and H and by calcium-activated neutral protease (that is, the enzymes sensitive to the inhibitors used). Because antipain, leupeptin and pepstatin significantly suppress such proteolysis, these agents might be useful in further assessing any potential contribution of cellular proteinases to the production of ischemic myocellular death. In addition, this study provides a new experimental model that affords serial assessments of regional myocardial proteolysis during the evolution of myocardial infarction.

Topics: Animals; Antipain; Cathepsins; Cell Survival; Chymotrypsin; Endopeptidases; Heart; Leupeptins; Male; Myocardial Infarction; Myocardium; Oligopeptides; Pepstatins; Protease Inhibitors; Rats; Rats, Inbred Strains; Time Factors; Tyrosine

Topics: Animals; Antipain; Cell Survival; Dose-Response Relationship, Drug; Drug Therapy, Combination; Endopeptidases; Heart; Leupeptins; Male; Myocardial Infarction; Myocardium; Pepstatins; Protease Inhibitors; Rats; Rats, Inbred Strains; Time Factors

n-Acetylneuraminic acid (NANA) was measured in the platelets and serum of normal subjects, patients with malignant disease, and patients with acute myocardial infarction. The malignant disease group had a mean platelet NANA 24% below normal and a serum NANA 68% above normal. Both results were highly significant. Samples from patients with acute myocardial infarction showed slightly reduced platelet NANA and slightly raised serum NANA, neither change being significant. There was no correlation between platelet and serum NANA in the neoplastic group.

Topics: Blood Platelets; Humans; Leupeptins; Myocardial Infarction; Neoplasms; Protease Inhibitors; Sialic Acids