Changes of the echocardiographic parameters in chronic heart failure patients with Ile337val, Glu23lys, and Ser1369ala polymorphisms of genes encoding the ATP-sensitive potassium channels subunits in the Ukrainian population.

Different allelic variants of genes that encode ATP-sensitive potassium (KATP ) channels' subunits may contribute to the development of heart failure. The purpose of the work to investigate SNPs in genes that encode KATP channels in relation to echocardiographic parameters in chronic heart failure (CHF) patients. Ninety-nine people with CHF of ischemic origin with left ventricular systolic dysfunction were examined. The control group is represented by 108 clinically healthy subjects. KCNJ11 polymorphisms Ile337Val and Glu23Lys, and ABCC8 polymorphism Ser1369Ala were genotyped using polymerase chain reaction. In CHF patients, the frequency of the Ile337Val genotype was: Ile/Ile, 40.4%; Ile/Val, 45.5%; and Val/Val, 14.1%. The patients with the Val/Val genotype had left ventricular (LV) mass that was 334.15 g, which was 27.3% (P < 0.05) lower versus Ile/Val patients (425.48 g). The index of this parameter was also significantly lower (28.4%, P < 0.05). In CHF patients, the frequency of Glu23Lys and Ser1369Ala was: Glu/Glu and Ser/Ser, 43.4%; heterozygote, 44.4%; Lys/Lys and Ala/Ala, 12.2%. The patients with the Lys/Lys and Ala/Ala genotypes had a significantly lower LV mass index and LV end-diastolic volume (22.9% and 26.8%, P < 0.05) versus heterozygotes. Thus, the greatest LV mass and LV end-diastolic volume values are associated with heterozygotes, while the smallest are associated with minor homozygotes.

Ageing related down-regulation of myocardial connexin-43 and up-regulation of MMP-2 may predict propensity to atrial fibrillation in experimental animals.

Mechanisms underlying atrial fibrillation (AF), the most common cardiac arrhythmia, particularly in aged population, are not fully elucidated. We have previously shown an increased propensity of old guinea pigs (GPs) heart to inducible AF when comparing to young animals. This study aimed to verify our hypothesis that susceptibility of aged heart to AF may be attributed to abnormalities in myocardial connexin-43 (Cx43) and extracellular matrix that affect cardiac electrical properties. Experiments were conducted on male and female 4-week-old and 24-week-old GPs. Atrial tissue was processed for analysis of Cx43 topology using immunohistochemistry, expression of Cx43 protein using immunobloting, and expression of mRNA of Cx43 and extracellular matrix metalloproteinase-2 (MMP-2) using real time PCR. Immunohistochemistry revealed uniform Cx43 distribution predominantly on lateral sides of the cardiomyocytes of young male and female GP atria. In contrast, non-uniform distribution, mislocalization and reduced immunolabeling of Cx43 were detected in atria of old GPs. In parallel, the atrial tissue levels of Cx43 mRNA were significantly decreased, while mRNA expression of MMP-2 was significantly increased in old versus young GPs. The changes were more pronounced in old GPs males comparing to females. Findings indicate that age-related down-regulation of atrial Cx43 and up-regulation of MMP-2 as well as disordered Cx43 distribution can facilitate development of AF in old guinea pig hearts.

siRNA-induced silencing of hypoxia-inducible factor 3α (HIF3α) increases endurance capacity in rats.

Molecular mechanisms of adaptation to exercise despite a large number of studies remain unclear. One of the crucial factors in this process is hypoxia inducible factor (HIF) that regulates transcription of many target genes encoding proteins that are implicated in molecular adaptation to hypoxia. Experiments were conducted on 24 adult male Fisher rats. Real-time PCR analysis was performed for quantitative evaluation of Hif3α, Igf1, Glut-4 and Pdk-1 in m. gastrocnemius, m. soleus, in lung and heart tissues. Mitochondrial respiratory function and electron microscopy were performed. Knockdown of Hif3α using siRNA increases time of swimming to exhaustion by 1.5 times. Level of mitochondrial NAD- and FAD-dependent oxidative pathways is decreased, however efficiency of phosphorylation is increased after Hif3α siRNA treatment. Expression of HIF target genes in muscles was not changed significantly, except for increasing of Pdk-1 expression in m. soleus by 2.1 times. More prominent changes were estimated in lung and heart: Igf1 gene expression was increased by 32.5 and 37.5 times correspondingly. Glut4 gene expression in lungs was increased from undetected level till 0.3 rel. units and by 84.2 times in heart. Level of Pdk1 gene expression was increased by 249.2 in lungs and by 35.1 times in hearts, correspondingly. Some destructive changes in muscle tissue were detected in animals with siRNA-inducing silencing of Hif3α.

Silencing of TERT decreases levels of miR-1, miR-21, miR-29a and miR-208a in cardiomyocytes.

To test the hypothesis that telomerase reverse transcriptase (TERT) as an RNA-dependent RNA polymerase could be involved in the amplification of microRNA (miRNA), we have determined the levels of immature and mature miRNA in cultured neonatal rat cardiomyocytes, during the silencing of TERT by siRNA. The silencing of the TERT gene led to the reduction of both telomerase activity and the TERT mRNA expression when compared with scrambled RNA. TERT gene silencing resulted in the decrement of three studied mature miRNAs levels: miRNA-21, miRNA-29a and miRNA-208a when compared with scrambled RNA; but miRNA-1, it was not changed significantly. At the same time, levels of immature miRNA-1 and miRNA-208a were not changed, although the levels of immature miRNA-29a and pri-miRNA-1 were decreased. The data obtained allow us to permit that TERT is a genome-independent source of mature miRNA, and the changes in telomerase activity can significantly influence the level of miRNA in cardiomyocytes.

[Influence of flocalin on development of apoptosis and necrosis at anoxia-reoxygenation of culture rats neonatal cardiomyocytes].

The influence of the new cardioprotector flocalin was investigated on the culture of rat's neonatal cardiomyocytes during anoxia-reoxygenation modelling. The mechanisms of apoptosis and necrosis were investigated under influence of ATP-sensitive potassium (K(ATP)) channels activation and in conditions of blocking of the L-type calcium (VGCCs) channels. Flocalin was added in the culture medium in the dose 5 and 20 microM at 2 minutes before anoxia (30 minutes) and following reoxygenation (60 minutes). These doses are near to: the first dose means the opening of K(ATP) channels and the second one means the IC50 block of VGCCs. It is discovered that in dose 5 microM of flocalin drew the change of correlation of living, necrotizing and apoptizing cells drew side-shifting living. The number of live cells was almost the same like in control (experiments without anoxia-reoxygenation modelling). The opening of K(ATP) channels decreases necrosis in two times and fully prevented development of apoptosis which was induced anoxia-reoxygenation modelling. Flocalin depressed the apoptosis of neonatal cardiomyocytes so that he was on to 36% less than in control group (without anoxia-reoxygenation). But in the high dose (20 microM) that provokes not only K(ATP) channels opening but also IC50 block of VGCCs cardioprotection was not detected after modelling of anoxia-reoxygenation. The last can be investigation both enough strong activating of the potassium channels and by investigation of both factors are opening of potassium and inhibition of VGCCs channels and, accordingly, substantial diminishing of level of introcellular Ca2+ and violation of metabolic processes yet to anoxia-reoxygenation. Thus, small doses of flocalin, that induce moderate opening of K(ATP) channels significantly decrease the number of necrotic and apoptotic cells in culture of rat neonatal cardiomyocytes induced by anoxia-reoxygenation.

Proteasome inhibitors eliminate protective effect of postconditioning in cultured neonatal cardiomyocytes.

A role of proteasomal proteolysis in the pathogenesis of ischemia-reperfusion is being actively studied. To evaluate the participation of the proteasome in postconditioning phenomenon, we used primary culture of neonatal cardiomyocytes. 30 minutes of anoxia followed by 60 minutes of reoxygenation was undergone. Postconditioning was modeled by 3 cycles of 1-minute reoxygenation followed by 1-minute anoxia, respectively. Clasto-lactacystin b-lactone, a specific proteasome inhibitor, in the dose that does not cause cell death (2.5 mM) was added to the culture medium just before the cycles of postconditioning. 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 led to an increase in trypsin-like and chymotrypsin-like activities comparing to anoxia, but these parameters never reached the control levels. PGPH activity was restored up to the initial level. 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 postconditioning. The data obtained permit to suppose that proteasome inhibitors can be used for pharmacological postconditioning.

[The influence of quercetin on the activity of purified 20S, 26S proteasome and proteasomal activity in isolated cardiomyocytes].

For the clarification of the effect of quercetin on the proteasome experiments were performed using purified 20S proteasome, 26S proteasome from the proteasomal fraction II (PF II), as well as cardiomyocyte culture which underwent anoxia-reoxygenation. In the experiments with purified 20S proteasome it was shown, that quercetin in a dose-dependent manner inhibits all three peptidase activities of the proteasome, comparable to a specific proteasome inhibitor. The highest quercetin inhibition was observed in the case of chymotrypsin-like activity of proteasome. In the same way quercetin inhibited the activity of 26S proteasome from the PF II. Quercetin decreased trypsin-like (by 26%, p = 0.03), chymotrypsin-like (by 63.7%, p = 0.04) and peptidyl-glutamyl peptide-hydrolyzing (by 34.2%, p = 0.16) activities in the cardiomyocytes culture. It appears, that quercetin and its water-soluble analogue korvitin affect the cardiomyocytes in the same manner, as specific proteasome inhibitors clasto-lactacystin-beta-lactone. In the concentrations 5 and 10 mM quercetin and korvitin resulted in the decrease of the amount of living cardiomyocytes, increasing the amount of necrotic and apoptotic cells. In the concentration 2.5 mM quercetin and korvitin significantly abolished damaging effect of anoxia-reoxygenation, decreasing the amount of necrotic and apoptotic cells. These data suggest that the mechanisms of cardioprotective effect of quercetin connected with inhibition of proteasome.

Postconditioning prevents apoptotic necrotic and autophagic cardiomyocyte cell death in culture.

In the paper the data concerning the possibility of the reproduction of the postconditioning phenomena in the cardyomicyte culture are presented. Primary cultures of cardiomyocytes from neonatal rats underwent 30 minutes of anoxia followed by 60 minutes of reoxygenation. Three different models of postconditioning were used: 3 cycles of 1, 3, or 5 minutes of reoxygenation followed by 1, 3, or 5 minutes of anoxia, respectively. The percentage of living, necrotic, and apoptotic cells were determined by staining with Hoechst 33342 and propidium iodide. Autophagy was demonstrated by the staining of vacuolar structures in vivo by monodansyl cadaverine. After anoxia and reoxygenation the amount of living, necrotic and apoptotic cells were 79 +/- 1.5, 7.8 +/- 0.9 and 13 +/- 1.5 %, respectively (in unstimulated cell culture 90 +/- 0.8, 3.3 +/- 0.3, and 5.5 +/- 0.7, P < 0.0001 for all). Postconditioning with 1 min anoxia 3-fold increased the amount of living cells and decreased the number of necrotic and apoptotic cells (P = 0.002, P = 0.02 and P = 0.043 respectively). Postconditioning with cycles of 3 and 5 minutes had a gradually reduced effect compared to cycles of 1 minute. The percentage of autophagic cells in control cell culture was 4.3 +/- 0.3%. This number increased after anoxia-reoxygenation to 14 +/- 0.8%, and was reduced by postconditioning (P < 0.001). The data obtained indicate that postconditioning is one of the effective methods of cardioprotection and could effectively decrease the amount of cardiomyocytes with traits of programmed or non-programmed cell death.

[Effect of N-3-methyladenine on peptidase and ribonuclease activity of proteasome].

Using a culture of cardiomyocytes it has been shown, that a well-known inhibitor of autophagy, N-3-methyladenine causes a 1.4 fold increase (p = 0.023) of the chymotrypsin-like activity, a 1.5 fold increase (p = 0.09) of the peptidyl-glutamyl peptide-hydrolyzing activity and 1.5 fold decrease (p = 0.07) of the trypsin-like activity of the proteasome. N-3-methyladenine in a dose-dependent manner inhibits chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of the purified 20S proteasome, but activates it trypsin-like activity. Chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of the 26S proteasome from proteasome fraction II did change in the same way, as in the case of 20S proteasome, but trypsin-like activity decreased. Using the above method of determining ribonuclease activity, we have shown, that N-3-methyladenine and clasto-lactacystin b-lactone inhibit the RNase activity of the proteasome. Specific proteasome inhibitor exhibits more powerful action, almost completely preventing RNA of actin and myosin from degradation. These data show a multitarget action of N-3-methyladenine, resulting in changes of peptidase and ribonuclease activity of the proteasome.

[Proteolytic enzymes and apoptosis]. / Proteoliticheskie fermenty i apoptoz.

Information about basic mechanisms of programmed cell death (apoptosis) development with participation of proteolytic enzymes is given in the review. The basic mechanisms of apoptosis launching are conditionally subdivided into three groups, depending on the "points of application" of apoptosis development initiating factor: membrane (receptor-dependent), mitochondrial and nuclear. Attention is accentuated on ubiquitin-dependent proteasomal proteolysis as the key system regulating apoptosis. The possible disturbances of apoptotic program realization are specified under various pathological processes and diseases.

[The diagnosis and treatment of complex forms of inguinal hernias]. / Diagnostika i lechenie slozhnykh form pakhovykh gryzh.

The volume pressure of the hydrodynamic stroke, and the "preservation" of the anterior and posterior walls of the inguinal canal were studied before and during operation in 100 patients with complex forms of inguinal hernia by means of devices suggested by the authors. With consideration for the obtained objective data, four degrees of severity of the disease were distinguished and the optimal methods for surgical correction were indicated.