Cardiovascular diseases represent the most important health risk factors because they are responsible for more than 50% of total mortality. Among them, the ischemic heart disease is leading cause of mortality. From the whole spectrum of different cardioprotective phenomena we have selected: 1) adaptation to chronic normobaric hypoxia (CNH) as the traditional experimental model in our laboratory area and 2) protective effect of exercise which in recent years represents promising and clinically relevant protective mechanism. The whole thesis is based on two studies. Aim of the first study was to characterize the expression of the main pro-inflammatory cytokine, TNF-α, in hearts of rats adapted to CNH. Chronic TNF-α inhibition by infliximab was used for discovering of certain role of TNF-α in CNH. We showed that increased myocardial level of TNF-α during adaptation to CNH was contributed via its receptor TNFR2 and nuclear factor κB-dependent activation of protective redox signalling with increased antioxidant defence. This adaptive pathway participates on the infarct size-limiting effect of CNH. Aim of the second study was find out whether exercise training and CNH could play synergy in cardiac protection in rats model. We reported that CNH and exercise reduced infarct size but their combination...

The aim of this thesis was to elucidate the role of carbonic anhydrase (CA) in the modulation of central pH/CO2-sensitive fictive breathing (measured using in vitro brainstem-spinal cord preparations) in leopard frogs (Rana pipiens) following exposure to chronic hypercapnia (CHC) and chronic hypoxia (CH). CHC caused an augmentation in fictive breathing compared to the controls (normoxic normocapnic). Addition of acetazolamide (ACTZ), a cell-permeant CA inhibitor, to the superfusate reduced fictive breathing in the controls and abolished the CHC-induced augmentation of fictive breathing. ACTZ had no effect on preparations taken from frogs exposed to CH. Addition of bovine CA to the superfusate did not alter fictive breathing in any group, suggesting that the effects of ACTZ were due to inhibition of intracellular CA. Taken together, these results indicate that CA is involved in central pH/CO2 chemoreception and the CHC-induced increase in fictive breathing in the leopard frog.

The aim of this thesis was to elucidate the role of carbonic anhydrase (CA) in the modulation of central pH/CO2-sensitive fictive breathing (measured using in vitro brainstem-spinal cord preparations) in leopard frogs (Rana pipiens) following exposure to chronic hypercapnia (CHC) and chronic hypoxia (CH). CHC caused an augmentation in fictive breathing compared to the controls (normoxic normocapnic). Addition of acetazolamide (ACTZ), a cell-permeant CA inhibitor, to the superfusate reduced fictive breathing in the controls and abolished the CHC-induced augmentation of fictive breathing. ACTZ had no effect on preparations taken from frogs exposed to CH. Addition of bovine CA to the superfusate did not alter fictive breathing in any group, suggesting that the effects of ACTZ were due to inhibition of intracellular CA. Taken together, these results indicate that CA is involved in central pH/CO2 chemoreception and the CHC-induced increase in fictive breathing in the leopard frog.

Les jonctions communicantes jouent un rôle important dans la prolifération et la réactivité vasculaire systémique. Dans cette étude, nous avons recherché le rôle de ces structures dans la paroi artérielle pulmonaire saine et dans l’hypertension artérielle pulmonaire (HTAP). L’HTAP est une pathologie de la circulation pulmonaire caractérisée par une prolifération cellulaire pathologique et une hyperréactivité à divers agonistes vasoconstricteurs tels que la sérotonine (5-HT). Dans la paroi artérielle pulmonaire de rats sains les protéines constituant les jonctions communicantes vasculaires (les connexines 37, 40 et 43) sont présentes. De plus, la connexine 43 située au niveau de la jonction myoendothéliale (JME) intervient dans la réactivité à la 5-HT. La 5-HT produit par ailleurs, (1) de l’anion superoxyde (O2?) au niveau du muscle lisse et (2) un vasodilatateur (le monoxyde d’azote (NO)) au niveau de l’endothélium. La JME va permettre le passage de l’O2? du muscle lisse vers l’endothélium de façon à piéger le NO et maintenir une contraction physiologique de l’artère pulmonaire. L’étude des jonctions communicantes dans la circulation pulmonaire pathologique a été réalisée sur deux modèles de rats atteints d’HTAP : (1) le modèle de rat atteint d’HTAP suite à une injection de monocrotaline (rat MCT) et (2) le modèle de rat atteint d’HTAP suite à une hypoxie chronique (rat HC). Les connexines 37, 40 et 43 sont également exprimées au niveau de la circulation pulmonaire de ces rats, mais leur distribution au niveau du muscle lisse et l’endothélium est modifiée. La réactivité des artères pulmonaires à divers agents vasoconstricteurs (5-HT, endothéline-1, phényléphrine, solutions dépolarisantes) est modifiée chez les rats MCT et HC. De plus, l’implication des jonctions communicantes dans la réactivité à ces agents est changée chez les rats MCT et HC. Ces données apportent de nouvelles connaissances sur le rôle des jonctions communicantes dans la réponse contractile des artères pulmonaires saines, et de nouveaux éléments permettant de mieux comprendre les altérations de la vasomotricité pulmonaire observée dans l’HTAP. / Gap junction communication plays an important role in proliferation and in the regulation of vascular reactivity. In this study, we investigated the role of gap junctions in physiological pulmonary circulation and in pulmonary arterial hypertension (PAH). PAH is the main pathology of pulmonary circulation and is characterized by cell proliferation and hyperreactivity to several contractile factors such as serotonin (5-HT). In pulmonary circulation from healthy rats, gap junction proteins were observed (connexins 37, 40 and 43). Moreover, connexin 43 located at the myoendothelial junction (MEJ) is involved in the reactivity to 5-HT in pulmonary arteries from healthy rats. Indeed, 5-HT produces (1) superoxyde anion (O2?) from smooth muscle and (2) a vasodilator (nitric oxide (NO)) from endothelium. O2? will pass through MEJ from the smooth muscle to the endothelium in order to scavenge NO and thus maintain contraction in physiological conditions. Gap junctions study in pathological pulmonary arteries has been performed on two animal models of PAH: (1) monocrotaline-induced PAH (MCT rat) and (2) chronic hypoxia-induced PAH (CH rat). Connexins 37, 40 and 43 were observed in pulmonary circulation from these rats, but the localization in smooth muscle and endothelium is modified. Moreover, pulmonary arterial vasoreactivity in response to several contractile agents (5-HT, endothelin-1, phenylephrine, depolarizing solutions) is modified in MCT and CH rats. Finally, the role of gap junctions in the reactivity to these agonists is changed in MCT and CH rats. These data have important implications for understanding physiological vasoreactivity to 5-HT in pulmonary circulation, and to better understand alterations of pulmonary arterial reactivity observed in PAH.

Au niveau des artères intrapulmonaires (AIP) de rats sains, la connexine 43 (Cx43) située au niveau de la jonction myoendothéliale (JME) intervient dans la réactivité à la 5-HT. La 5-HT produit de l’anion superoxyde (O2•) au niveau du muscle lisse et du monoxyde d’azote (NO) au niveau de l’endothélium des AIP. La Cx43 permet alors le passage de l’O2• du muscle lisse vers l’endothélium de façon à diminuer la biodisponibilité du NO et maintenir une contraction physiologique de l’artère pulmonaire. Cependant, l’augmentation d’O2• par d’autres agonistes vasoconstricteurs tels que l’endothéline-1 (ET-1) et la phényléphrine (PHE) et les mécanismes impliqués dans cette augmentation restent encore méconnus dans la circulation pulmonaire. Ainsi, ce travail vise à identifier les agonistes qui provoquent une augmentation de la quantité d’O2• et à mettre en évidence les voies de signalisation mises en jeu au niveau des AIP de rats. Par ailleurs, la Cx43 étant impliquée dans la réactivité des AIP de rats sains à la 5-HT, le rôle de la Cx43 dans la circulation pulmonaire pathologique a été étudié à l’aide d’un modèle de souris hétérozygote pour la Cx43 (souris Cx43+/-) souffrant d’hypertension pulmonaire (HTP) hypoxique chronique (HC). Nous avons montré que l’augmentation d’O2•au niveau des AIP est un mécanisme exclusif de la 5-HT. Cette augmentation provient uniquement d’une augmentation de production par la mitochondrie et les NADPH oxydases via un mécanisme de « ROS-induced ROS release » dépendant de la PKC. La 5-HT agit par le biais des récepteurs 5-HT2A, induit un influx calcique extracellulaire qui augmente la concentration calcique mitochondriale provoquant une augmentation de production d’O2• par le complexe I de la chaîne respiratoire mitochondriale. De plus, cette voie s’exerce au sein des microdomaines de signalisation, notamment les cavéoles. D’autre part, chez les souris Cx43+/- HTP HC le remodelage des AIPest inhibé et l’hypertrophie ventriculaire droite est freinée. La vasoréactivité des AIP à la 5-HT, à l’ET-1 et à la PHE est modifiée chez les souris Cx43+/- saines et HTP HC par rapport aux souris sauvages. Ces données apportent (1) des éléments de compréhension sur les voies de signalisation mises en jeu dans la production d’O2• en réponse à la 5-HT dans la circulation pulmonaire de rats sains et (2) de connaissances nouvelles sur le rôle de la Cx43 dans les AIP de souris saines et souffrant d’HTP HC. / Under physiological conditions, in rat intrapulmonary arteries (IPA), connexin 43 (Cx43) localised at the myoendothelial junctions is involved in the reactivity to serotonin (5-HT). 5-HT increases superoxide anion (O2•) in the smooth muscle and nitric oxide (a vasodilator) in the endothelium. O2• will then rapidly pass through Cx43 to scavenge endothelial NO to decrease the bioavailability of the latter and hence maintain IPA contraction in physiological conditions. However, to date, the increase in O2• level by other contractile agonists such as endothelin-1 (ET-1) and phenylephrine (PHE) and the mechanism involved in this increase are still unknown in the pulmonary circulation. The goal of this present work is to identify the contractile agonists involved in the increase of O2• and to highlight the signaling pathways involved in the agonists-induced increase of O2•. As Cx43 plays an important role in IPA contractile reactivity to 5-HT in healthy rats, the role of Cx43 under pathological conditions of the pulmonary circulation has been studied with a mice model heterozygous for Cx43 (Cx43+/-) suffering from hypoxic pulmonary hypertension (HPH). We have shown that O2• increase in rat IPA is exclusive to 5-HT. 5-HT-induced O2• increase in rat IPA is only from an increase in production by the mitochondria and NADPH oxydase via a ROS-induced ROS release mechanism dependent on PKC. Upon binding to 5-HT2A receptors, 5-HT induces an extracellular calcium influx which is responsible for an increase in mitochondrial calcium level and causes an upregulation in O2• production by the complex I of the mitochondrial respiratory chain. Moreover, this signaling pathway takes place in specialized microdomains, namely caveolae. On the other hand, interestingly, in Cx43+/- HPH mice, IPA remodeling is inhibited and right ventricular hypertrophy is less intense. IPA vasoreactivity to 5-HT, ET-1 and PHE is modified in Cx43+/- healthy and HPH mice as compared to wild type mice. These data bring (1) new elements of comprehension in the signaling pathways involved in 5-HT-induced O2• production in healthy rat pulmonary circulation and (2) new insights on the role of Cx43 in mice IPA under physiological and HPH conditions.

- 4 - Abstract Adaptation to hypoxia or exercise training has cardioprotective effects against acute ischemic injury, but can potentially negatively influence heart function. Possible negative changes depend on the degree of hypoxia and exercise training intensity. It is therefore necessary to evaluate the effects of the specific adaptation protocols used. The ideal technique is echocardiography, which enables non-invasive, repeated and long-term measurements of the same individual allowing to study the development of changes in the course of adaptation. The aim of this study was to determine the effects of selected protocols of adaptation to intermittent hypobaric hypoxia (corresponding to the altitude of 4,000 to 8,000 meters above sea level, for 15 weeks in total) and exercise training (running speed 30 m.min-1 for 60 min a day, 4 weeks in total) on the left ventricle geometry and systolic function in rats. We assessed basic echocardiographic parameters of the ventricle geometry and function such as fractional shortening, ejection fraction, stroke volume, cardiac output etc. The adaptation of rats to intermittent hypobaric hypoxia lead neither to the impairment of systolic function nor to the development of left ventricle hypertrophy compared to controls; signs of moderate hypertrophy were observed only...

Cardiovascular diseases, particularly acute myocardial infarction, are the leading causes of death in developed countries including the Czech Republic. One of the ways to increase cardiac resistance against acute ischemia/reperfusion (I/R) injury is adaptation to chronic hypoxia. However, changes at the molecular level associated with this adaptation have still not been fully explored. It is obvious that the myocardial function depends on maintaining membrane integrity and cellular homeostasis of cardiomyocytes. From this perspective, phospholipases A2 (PLA2) are the key enzymes that take part in the remodeling and repairing of the cell membranes. Moreover, PLA2 are also involved in generation of lipid signaling molecules - free long chain fatty acids (FA) and 2-lysophopholipids. In myocardium, members of three major PLA2 classes are present: cytosolic PLA2 (cPLA2), calcium-independent PLA2 (iPLA2) and secretoric PLA2 (sPLA2). This thesis aimed to determine the following in the left ventricular myocardium of adult male Wistar rats: 1) The effect of intermittent hypobaric hypoxia (IHH; 8 hours/day, 5 days/week, 5 weeks, ~ 7000 m) on the expression of total cPLA2α and its phosphorylated form (p-cPLA2α, Ser505 ), and further iPLA2 and sPLA2IIA, as well as signaling proteins activating cPLA2α enzyme...

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...