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Myocardial tolerance to ischemia/reperfusion injury - possible protective mechanisms
Alánová, Petra ; Neckář, Jan (advisor) ; Nováková, Olga (referee) ; Vaněčková, Ivana (referee)
Ischemic heart disease is the leading cause of death and disability worldwide. The effects of ischemic heart disease are usually attributable to the detrimental effects of acute myocardial ischemia/reperfusion (I/R) injury. The aim of the thesis was to contribute to current effort to clarify the basis of mechanisms that could save the heart from I/R injury. The whole thesis is based on four studies; while the first three are published, the fourth one has been under revision. In the first study, we proved the involvement of nitric oxide (NO) in the cardioprotective mechanism of chronic hypoxia (CH). We described that exogenously increased availability of NO as well as inhibition of phosphodiesterase type 5 led to increased myocardial tolerance of normoxic and chronically hypoxic rats. The effects of both interventions were not additive, suggesting that NO is included in cardioprotective signaling of CH. Second study continued in investigating molecular mechanisms underlying cardioprotection induced by CH. We showed that infarct size-limiting effect of adaptation to CH was accompanied by increased myocardial concentration of tumor-necrosis factor alpha (TNF-α) and TNF-α receptor R2. In the third study, we examined the effect of dexrazoxane (DEX), the only clinically approved drug against...
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Mitochondrial respiration at cold acclimated rats. Comparison of tissues.
Flégrová, Eliška ; Žurmanová, Jitka (advisor) ; Nováková, Olga (referee)
Acclimation to cold or hardening is known for many decades through its beneficial effects on human health. In contrast, sudden exposure to cold, cold shock, is a great risk of cerebral and cardiac injury, especially in the elderly. There is very little published data on the cellular and molecular mechanisms induced by cold adaptation in heart and brain. The aim of this work was to describe and compare different properties heart, liver, brain and brown adipose tissue mitochondria of rats housed at 25 ± 1 řC and at mild cold (9 ± 1 řC, 5 weeks). The high-resolution oxygraphy, spectrophotometry and Western blotting analyses were used. We found differences in the respiratory control between the heart and liver. Cold acclimation decreased activity of the Krebs cycle enzymes. Fatty acid contribution to the respiration reached the maximum in brown fat and the minimum in the hippocampus. However, further study is necessary.
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Compartmentalization of the beta-adrenergic signaling system in cardiac cells: the effect of hypoxia
Karlovská, Ivana ; Novotný, Jiří (advisor) ; Nováková, Olga (referee)
The aim of this thesis was to study the changes that occur in cell line H9c2 after exposure to an oxygen level reduced to 2 % for 24 hours. We monitored changes in compartmentation of chosen members of β-adrenergic signaling system. We found an increase in expression of β1AR and β2AR. Only β2AR showed change in compartmentation after hypoxia, as they relocate from membrane rafts to non-rafts fractions of membrane. AC also showed an increase of expression and was located in membrane rafts. The next aim of this work was to monitore apoptotic markers to determine whether there are activated pro-apoptotic or anti-apoptotic signals under chosen conditions of hypoxia. There was an increase in expression of both pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2. We compare ratios of Bcl-2 to Bax and we found that there is a bigger increase in protein Bax expression. Another apoptotic marker, caspase 3, was tested and we also found that there was an increase in expression of caspase 3 in cells after hypoxia. Furthermore, we studied possible activation of kinase signaling pathways that may contribute to protective effects of hypoxia. Expression of Akt and ERK kinases was increased after hypoxia, but we did not confirm activation by phosphorylation of these kinases. Levels of phosphorylated Akt...
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Gene expression of selected genes in heart of long-term cold acclimated rat under mild conditions
Marvanová, Aneta ; Žurmanová, Jitka (advisor) ; Nováková, Olga (referee)
Beneficial effect of hardening or cold adaptation for human health has been known for many decades. Beneficial or detrimental effect depends on the regimen and intensity of the adaptation and thus it is very important to pay attention to them. It has been shown in previous studies that cold adaptation of animals at a low temperature 0-4 žC is accompanied by dehydration and the damage of kidneys. As our best knowledge, the effect of mild cold adaptation on the molecular mechanisms in the heart has not been studied until now. The first aim of this study was to set up a new model of cold adaptation without any negative effects on the organism. The second aim was to assess mRNA levels of selected genes related to the important mechanisms associated with energy metabolism and cardio-protective signaling pathways in the left ventricle. Results obtained using the new model for cold adaptation at 102 řC did not show any negative changes in the blood or urine parameters and body fluid balance was maintained. Screening of mRNAs showed that chronic adaptation might affect the phenotype of the heart, transcriptional activity of the antioxidant system and of the pro-apoptotic genes, lowers adrenergic signaling and moderate glucose metabolism in cardiomyocytes. Key words: cold adaptation, heart, gene...
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A study of adenosine receptors and their signaling in the rat myocardium
Eichlerová, Lenka ; Novotný, Jiří (advisor) ; Nováková, Olga (referee)
Adenosine plays a critical role in the heart signalling while affecting heart rate, contractility or coronary flow. Nowadays, four adenosine receptor subtypes are distinguished which are present in most of tissues and cells: A1, A2A, A2B and A3. All these receptors belong to the family of G protein-coupled receptors. Upon activation, their main target is an enzyme adenylyl cyclase which produces an important second messenger cAMP. The main goal of this thesis was characterization of adenosine receptors in the rat myocardium, assessment of their distribution, binding properties and signalling. We examined a possible disparity in receptors distribution between the left and right ventricles using SDS-PAGE electrophoresis and Western blotting. The same methods have been used in studies of adenosine receptor distribution in lipid rafts. Samples of lipid rafts and soluble fraction were prepared using a nonionic detergent Triton X-100. We did not find any evidence of different distribution between the left and right ventricles and our results did not confirm compartmentation of the receptors either. For determination of binding properties of the receptors we used radioligand binding assays with the A1 selective radioligand [H3 ]DPCPX. We did not observe any significant difference between the receptor...
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The role of mitochondrial creatine kinase and hexokinase in cardioprotective mechanisms induced by chronic hypoxia
Wasková, Petra ; Žurmanová, Jitka (advisor) ; Drahota, Zdeněk (referee) ; Nováková, Olga (referee)
IN ENGLISH The ischemia-reperfusion (I/R) injury, which is a consequence of myocardial infarction, represents a major cause of death worldwide. One of the most effective cardioprotective interventions increasing the resistance of hearts to the I/R injury is the adaptation to a chronic hypoxia (CH). However, the molecular mechanisms of CH are still not well understood. The most important factors responsible for the I/R injury are reactive oxygen species (ROS) produced by complexes I and III within the mitochondrial electron transport chain. Potential candidates maintaining ROS at a low level are mitochondrial creatine kinase (mtCK) and two hexokinase isoforms (HK1 and HK2). These enzymes highly support the mitochondrial oxidative phosphorylation by increasing the availability of ADP for complex V of the respiratory chain. In addition, the HK binding to mitochondria inhibits binding of the pro- apoptotic protein BAX, thereby protecting cardiac cells against apoptosis. Besides the mitochondrial CK isoform, there are two cytosolic CK (CKM and CKB) present in cardiomyocytes that help to maintain energy homeostasis. Based on the known anatomical and physiological differences between the left (LV) and the right (RV) ventricles, the first study focused on the comparing ventricles in terms of the energy...
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Effect of chronic hypoxia on antioxidative capacity of rat myocardium.
Závišková, Kristýna ; Nováková, Olga (advisor) ; Žurmanová, Jitka (referee)
Adaptation to chronic hypoxia activates endogenous signaling cascades, which lead to cardiac protection against acute ischemia/reperfusion (I/R) injury. The molecular mechanism of this phenomenon has not been fully clarified yet. However, it was proved that reactive oxygen species (ROS) take part in cardioprotective signaling pathway inducted by chronic hypoxia. The high level of ROS must be precisely regulated by antioxidative system of a cell. The aim of diploma thesis was to examine the effect of intermittent hypobaric hypoxia (IHH, 7 000 m) on relative amount of antioxidative enzymes (peroxiredoxin 6 - PRX6, thioredoxin 1 and 2 - TRX1 and TRX2, thioredoxin reductase 1 - TRXR1) and also enzymes of iron metabolism (heme oxygenase 1 and 2 - HO1 and HO2, aconitase 1 and 2 - ACO1 and ACO2), which participate in regulation of cell redox state. Moreover, we studied the effect of adaptation to IHH and an antioxidant tempol on relative amount of calcium-independent phospholipase A2 (iPLA2). iPLA2 can remove peroxidized fatty acids from membrane phospholipids. On the other hand, iPLA2 can damage cell in I/R conditions. All enzymes were studied in homogenates from normoxic and IHH adapted rat left ventricular myocardium by Western blot. Adaptation to IHH caused a decrease of PRX6 and on the opposite an increase of...
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Analysis of PKCα Influence on Cancer Cell Invasion.
Szabadosová, Emília ; Brábek, Jan (advisor) ; Nováková, Olga (referee)
7 Abstract Protein kinase C alpha (PKCα) is a serine/threonine protein kinase. PKCα is an important protein regulating cell polarity, protein secretion, apoptosis, cell proliferation and differentiation and tumorogenesis. Previous research has shown a role of PKCα also in a cancer cell migration and cancer cell invasion. The aim of this study was to investigate the role of protein kinase C alpha (PKCα) played in amoeboid mode of cancer cell invasion. We showed that higher expression of PKCα resulted in mesenchymal-amoeboid transition of K2 and MDA mesenchymal cancer cell lines, which was accompanied with decreased cancer cell invasive capability in 3D collage matrix. PKCα overexpression had no effect on the cell morphology of A375m2, however, the results showed a trend in increased invasive potential of A375m2 cells. Conversely, the expression of dominant-negative PKCα resulted in amoeboid-mesenchymal transition of A375m2 cells, and it was associated with decreased invasive potential of K2 and MDA cell lines. Furthermore, a linkage between PKCα and phosphatidylinositol 3-kinase (PI3K) was tested. The results revealed that increased activity of PKCα was accompanied with decreased level of active Akt in K2 cell line. To summarize, our results suggest a probable role of PKCα in regulation of amoeboid...
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The role of sphingosin kinase in cardioprotection
Pospíšilová, Barbora ; Nováková, Olga (advisor) ; Kolář, David (referee)
Sphingosine-1-phosphate (S1P) is bioactive mediator with cardioprotective effect. Sphingosine kinase (SK) is a key enzyme in the synthesis of S1P. It exists in the two isoforms sphingosine kinase 1 and sphingosine kinase 2. Although SK1 has antiapoptotic feature and SK2 has proapoptotic feature both are crucial for the effect of S1P. S1P can act and affect the cellular faith by intracellular or extracellular functioning. Extracellular S1P binds and activates specific cell surface receptors on the plasma membrane. These receptors are members of the group of G protein-coupled receptors. There are tree subtypes of S1P receptors in the heart tissue (S1P1, S1P2, S1P3). Exogenous S1P increases viability of cardiomyocytes after myocardiacal ischemia/reperfusion injury (I/R). It also reduces the infarct size in isolated rat heart. During conditions of ischemic preconditioning (IPC) or postconditioning (IPOST) which consist of the short periods of ischemia before or after major ischemia insult generate S1P. Released S1P increased viability of the cell and faster recovery of hemodynamic functions in the heart tissue. Effects of S1P and its role in cardioprotection are explored in the genetically modified organisms mainly in the mouses. The evidence of the cardioprotective effect of S1P were experiments using...
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Role of phospholipases A2 in cardioprotection induced by continuous normobaric hypoxia in rat heart
Kyclerová, Eva ; Nováková, Olga (advisor) ; Novotný, Jiří (referee)
Recently, they are examined various means for activating the endogenous signalling pathways leading to increased resistance of the myocardium from ischemic/reperfusion (I/R) injury. One of them is the adaptation to chronic hypoxia, which has been shown to reduce the incidence and severity of ventricular arrhythmias, improves the recovery of postischemic contractile function of the heart and particularly reduces the extent of myocardial infarction. Since the function of the heart depends on the maintenance of membrane integrity of cardiomyocytes there are very important phospholipase A2 (PLA2) which are involved in the repair of cellular membranes. Also they are an important component of the protective signalling pathways because they cleave membrane phospholipids to produce lipid signalling molecules. Elucidate the role of PLA2 and the precise mechanism of action of signalling pathways leading to cardioprotection could be important for the prevention and treatment of cardiovascular diseases. Therefore, in this thesis we examined the influence of continuous normobaric hypoxia (CNH) to the relative representation of cardiac PLA2 (secretory - sPLA2IIA, calcium-independent - iPLA2, cytosolic - cPLA2α and its phosphorylated form - p-cPLA2α), and proteins involved in the activation and phosphorylation of...
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