Proteasomal proteolysis in anoxia-reoxygenation, preconditioning and postconditioning of isolated cardiomyocytes.

The role of proteasomal proteolysis in the pathogenesis of ischemia-reperfusion is being actively studied. To evaluate the participation of the proteasome in the preconditioning and postconditioning phenomena we used primary culture of neonatal cardiomyocytes. This culture was undergone 30min of anoxia followed by 60min of reoxygenation. Preconditioning was modeled by three cycles of 3min anoxia followed by 3min reoxygenation. Postconditioning was modeled by three cycles of 1min reoxygenation followed by 1min anoxia, respectively. Clasto-lactacystin beta-lactone, a specific proteasome inhibitor, was added to the culture medium right before the cycles of preconditioning or postconditioning in the dose that does not cause cell death (2.5muM). Percentages of living, necrotic, and apoptotic cells were determined by staining with bisbenzimide and propidium iodide. Autophagy was demonstrated by staining vacuolar structures with monodansyl cadaverine. Proteasomal activity was determined by cleavage intensity of specific fluorogenic substrates. Trypsin-like, chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities were decreased after anoxia. Reoxygenation has led to the increase in trypsin-like and chymotrypsin-like activities comparing to anoxia, but these parameters have never reached the control levels. PGPH activity has been restored up to the initial level. Preconditioning and postconditioning increased numbers of living cells and decreased that of necrotic, apoptotic and autophagic cells. Paradoxically, it was established that proteasome inhibitors prevented the necrotic and apoptotic cell death of cardiomyocytes in anoxia-reoxygenation, but in the same concentration abolished the effects of preconditioning and postconditioning. Low doses of proteasome inhibitors, particularly the ones used in our experiments, resulted in the abolishing of preconditioning and postconditioning phenomena, but at the same time led to the increase of the population of living cells in anoxia-reoxygenation, and can be considered as potential pharmacological agents of preconditioning and postconditioning.

Allelic polymorphism in the promoter (T-->C), but not in exon 7 (G-->T) or the variable number tandem repeat in intron 4, of the endothelial nitric oxide synthase gene is positively associated with acute coronary syndrome in the Ukrainian population.

BACKGROUND: Different allelic variants of endothelial nitric oxide synthase (eNOS) can have different effects on the development of certain forms of ischemic heart disease, depending on the specific human population. The frequency of the polymorphism T(-786) --> C in the promoter, G(894)-->T in exon 7 and the variable number tandem repeat in intron 4 were assayed in patients with acute coronary syndrome compared with clinically healthy individuals in the Ukrainian population. METHODS: Polymerase chain reaction and restriction fragment length polymorphism analysis were used to detect the above mentioned variants of the eNOS gene in 221 patients with acute coronary syndrome and in 83 control subjects. RESULTS: It was shown that the percentage of normal homozygotes, heterozygotes and pathological homozygotes for the T(-786)-->C promoter polymorphism was 47.5%, 36.2% and 16.3%, respectively (controls: 48.2%, 45.8% and 6.0%; P<0.05 by chi(2) test); for the G(894)-->T polymorphism in exon 7, the percentages were 34.4%, 57.9% and 7.7%, respectively (controls: 28.9%, 67.5% and 3.6%; P>0.05); and, for the 4a/4b polymorphism in intron 4, the percentages were 64.7%, 31.2% and 4.1%, respectively (controls: 62.7%, 32.5% and 4.8%; P>0.05). CONCLUSIONS: The C/C promoter variant of eNOS can be considered a risk factor for acute coronary syndrome in the Ukrainian population.