Chronic Hypoxia Induces Epigenetic Modifications in the Fetal Rat Heart

Heart disease remains the leading cause of death worldwide. As a result of studies done by Barker and associates, our awareness of the significance of stress during gestation as a risk factor for heart diseases has expanded. We now know that events in utero can significantly alter gene expression patterns in heart tissue leading to increase susceptibility to ischemia reperfusion injury in adulthood. The focus of this project was to elucidate the role of chronic hypoxia in the programming of the cardio-protective gene, Protein Kinase C epsilon (PKCε) in fetal rat heart. We used an animal, organ base, and cell culturing with the rat embryonic cell line H9c2 to determine the molecular events underpinning the heightened sensitivity to ischemia reperfusion injury of adult offspring exposed to chronic hypoxia in utero. We determined that chronic hypoxia directly represses PKCε expression through increase methylation of CpG dinucleotides for two SP1 binding sites located at proximal region of the PKCε promoter. Previous studies using reporter gene assays concluded the region encompassing both SP1 binding sites played a significant role in the activity of PKCε promoter. Chromatin immunoprecipitation (ChIP) assays further verified the functional significance of methylation for both Sp1 sites in reducing SP1 protein binding. In the presence of the DNA methylation inhibitor, 5-aza-2-deoxycytidine, binding of SP1 to PKCε promoter, promoter methylation, and PKCε protein and mRNA were restored to control levels. Connecting epigenetics to chronic hypoxia in utero led us to further investigate the underlining mechanism of hypoxia-induced methylation of PKCε promoter. The dominant pathway of cellular adaptation to hypoxic stress involves the stabilization of the Hypoxia-Inducible-Factor 1 alpha (HIF-1α). We found blockade of nuclear accumulation of HIF-1α did not restore PKCε mRNA to control values. Next, we found the ROS Scavengers N-acetylcysteine and 4-hydroxy Tempo protect against hypoxia-induced repression of PKCε gene activity, which linked oxidative stress to PKCε repression in fetal hearts. This project has demonstrated that chronic hypoxia directly regulates PKCε gene expression through ROS mediated epigenetic repression of PKCε promoter, which leads to long term programming of the fetal heart

The role of protein kinase C and its targets in cardioprotection

The mortality of cardiovascular diseases remains high and it likely tends to increase in the future. Although many ways how to increase the resistance against myocardial ischemia- reperfusion damage have been described, few of them were transferred into clinical practice. Cardioprotective effect of chronic hypoxia has been described during 60s of the last century. Its detailed mechanism has not been elucidated, but a number of components has been identified. One of these components presents protein kinase C (PKC). The role of PKC was described in detail in the mechanism of ischemic preconditioning, but its involvement in the mechanism of cardioprotection induced by chronic hypoxia remains unclear. One reason is the amount of PKC isoforms, which have often contradictory effects, and the diversity of hypoxic models used. The most frequently mentioned isoforms in connection with cardioprotection are PKCδ and PKCε. The aim of my thesis was to analyze changes in these PKC isoforms at two different cardioprotective models of hypoxia - intermittent hypobaric (IHH) and continuous normobaric hypoxia (CNH). We also examined the target proteins of PKCδ and PKCε after the adaptation to IHH, which could be involved in the mechanism of cardioprotection. These included proteins associated with apoptosis and...Mortalita kardiovaskulárních onemocnění je stále vysoká a pravděpodobně bude mít v budoucnu tendenci se spíše zvyšovat. Přestože byla popsána řada způsobů, jak odolnost myokardu vůči ischemicko-reperfúznímu poškození zvýšit, minimum z nich bylo přeneseno do klinické praxe. Kardioprotektivní působení chronické hypoxie bylo popsáno již v 60. letech minulého století. Jeho detailní mechanismus nebyl doposud objasněn, ale byla identifikována řada komponent, které se zde uplatňují. Jednou z nich je proteinkinasa C (PKC). Úloha PKC byla podrobně popsána v mechanismu ischemického preconditioningu, její zapojení v mechanismu kardioprotekce vyvolané hypoxií však zůstává nejasné. Jedním z důvodů je množství isoforem PKC, které mají mnohdy protichůdné účinky, a také různorodost používaných hypoxických modelů. V souvislosti s kardioprotekcí jsou nejčastěji zmiňovány isoformy PKCδ a PKCε. Cílem mé práce bylo analyzovat změny těchto isoforem PKC na dvou různých kardioprotektivních modelech hypoxie - intermitentní hypobarické (IHH) a kontinuální normobarické hypoxii (CNH). Zároveň jsme po adaptaci na IHH sledovali cílové proteiny PKCδ a PKCε, které by mohly být do mechanismu kardioprotekce zapojeny. Jednalo se o proteiny spojené s apoptosou a autofagií, s dynamikou mitochondrií, odstraňováním toxických aldehydů,...Department of Cell BiologyKatedra buněčné biologieFaculty of SciencePřírodovědecká fakult

Cardioprotective Regimen of Adaptation to Chronic Hypoxia Diversely Alters Myocardial Gene Expression in SHR and SHR-mtBN Conplastic Rat Strains

Adaptation to continuous normobaric hypoxia (CNH) protects the heart against acute ischemia/reperfusion injury. Recently, we have demonstrated the infarct size-limiting effect of CNH also in hearts of spontaneously hypertensive rats (SHR) and in conplastic SHR-mtBN strain characterized by the selective replacement of the mitochondrial genome of SHR with that of more ischemia-resistant Brown Norway rats. Importantly, cardioprotective effect of CNH was more pronounced in SHR-mtBN than in SHR. Thus, here we aimed to identify candidate genes which may contribute to this difference between the strains. Rats were adapted to CNH (FiO2 0.1) for 3 weeks or kept at room air as normoxic controls. Screening of 45 transcripts was performed in left ventricles using Biomark Chip. Significant differences between the groups were analyzed by univariate analysis (ANOVA) and the genes contributing to the differences between the strains unmasked by CNH were identified by multivariate analyses (PCA, SOM). ANOVA with Bonferroni correction revealed that transcripts differently affected by CNH in SHR and SHR-mtBN belong predominantly to lipid metabolism and antioxidant defense. PCA divided four experimental groups into two main clusters corresponding to chronically hypoxic and normoxic groups, and differences between the strains were more pronounced after CNH. Subsequently, the following 14 candidate transcripts were selected by PCA, and confirmed by SOM analyses, that can contribute to the strain differences in cardioprotective phenotype afforded by CNH: Alkaline ceramidase 2 (Acer2), Fatty acid translocase (Cd36), Aconitase 1 (Aco1), Peroxisome proliferator activated receptor gamma (Pparg), Hemoxygenase 2 (Hmox2), Phospholipase A2 group IIA (Ppla2g2a), Dynamin-related protein (Drp), Protein kinase C epsilon (Pkce), Hexokinase 2 (Hk2), Sphingomyelin synthase 2 (Sgms2), Caspase 3 (Casp3), Mitofussin 1 (Mfn1), Phospholipase A2 group V (Pla2g5), and Catalase (Cat). Our data suggest that the stronger cardioprotective phenotype of conplastic SHR-mtBN strain afforded by CNH is associated with either preventing the drop or increasing the expression of transcripts related to energy metabolism, antioxidant response and mitochondrial dynamics

Functional proteomic analysis of the myocardial PKCepsilon subproteome PKCepsilon-AKT-eNOS signaling modules and during.

Ischemic heart disease is the leading cause of mortality and morbidity in Western societies. Thus, understanding the molecular mechanisms to reduce myocardial ischemia and limit infarction size are of great importance. Ample evidence has shown that protein kinase C epsilon (PKCepsilon) play an essential role in the genesis of cardioprotection. In particular, our laboratory has shown that activation of PKCepsilon in the heart is sufficient to significantly reduce myocardial infarction due to coronary artery occlusion. However, the molecular mechanism responsible for PKCepsilon-induced cardioprotection remain unclear. Recently, functional proteomic analysis have demonstrated that PKCepsilon forms signaling complexes with various of proteins, including structural proteins, signaling molecules, stress-activated proteins, transcription al/translational factors, metabolism-related proteins, and PKC-binding domain containing proteins, and the coordination of these molecules contributes to cardioprotection. Despite this information which obtained from whole heart lysates, little is known regarding the cellular mechanisms that regulate the assembly of these protein complexes and the specific manner in which these molecules interact with each. Therefore, this dissertation is focused on the characterization of PKCepsilon subproteome and signaling modules within, as well as their regulation during cardioprotection. PKCepsilon-Akt-eNOS signaling module is chosen as my research paradigm because the individual molecule PKCepsilon, Akt and eNOS has been implicated in the prevention of cell death. In addition, both eNOS and Akt were found to exist in the PKCepsilon complex in our initial studies, suggesting that functional coupling among these molecules may be a heretofore unrecognized protective signaling mechanism. Our data demonstrate that the cardiac PKCepsilon signaling subproteome is comprised of various sizes of protein complexes, and the formation of these complexes and modules is regulated by the subcellular localization and extracellular stimulus. During cardioprotection, activation of PKCepsilon enhances the PKCepsilon-Akt-eNOS module formation, and thus contributes to the regulation of nitric oxide production and hence the manifestation of the cardioprotective phenotype. In conclusion, the present research successfully characterizes the myocardial PKCepsilon subproteome and PKCepsilon-Akt-eNOS signaling modules, which serves as an important step towards our complete understanding of the signaling mechanisms underlying PKCepsilon-mediated card ioprotection and ultimately aids in the development of pharmacological agents to protect the heart

Studies on the role of connexin 43 phosphorylation in the injury - resistant heart

Ischemic heart disease is a major cause of death worldwide. Identifying the mechanisms mediating cardiac resistance to injury (‘cardioprotection’) can contribute to therapies for cardiac injury. These studies investigated the role of the membrane channel forming protein connexin43 (Cx43), a downstream effector of PKCε-mediated cardioprotection. In isolated cardiomyocytes FGF-2/PKCε –induced cytoprotection is mediated by Cx43 phosphorylation at PKCε-target site, serine(S)262. Hypothesis (1). PKCε-mediated cardioprotection increases Cx43 phosphorylation at PKCε-target sites and prevents ischemia and/or reperfusion-induced Cx43 remodeling. Rat hearts were subjected to protective treatments (ischemic preconditioning, FGF-2, diazoxide), followed by 30 min global ischemia and/or 60 min reperfusion. All treatments elicited above-physiological levels of phospho(p)S262-Cx43 and pS368-Cx43 (P*Cx43), which were sustained during global ischemia, and reperfusion, and accompanied by attenuation of ischemia-induced Cx43 dephosphorylation, and prevention of Cx43 lateralization. Cx43 is present in cardiac subsarcolemmal (SSM) but not interfibrillar (IFM) mitochondria. Hypothesis (2). FGF-2 exerts protective effects on both mitochondrial populations, but is associated with mitochondrial (mito) P*Cx43 state in SSM, and mediated by mitoCx43 function. FGF-2 treatment increased calcium tolerance in SSM and IFM by 2.9- and 1.7-fold, respectively, compared to controls. In the presence of Gap27, a Cx43 hemi-channel blocker, the salutary effect of FGF-2 were lost in SSM but not IFM, indicating a functional role for Cx43. FGF-2 increased levels of PKCε, pPKCε and Tom-20 translocase in SSM and IFM. In SSM, FGF-2 increased pS262-and pS368-Cx43 by 30-fold and 8-fold, compared to controls. Stimulation of untreated SSM with a PKC activator (phorbol 12-myristate 13-acetate; PMA) also increased pS262-and-S368-Cx43 and calcium tolerance, which was prevented by εV1-2, a PKCε-inhibiting peptide. The effect of FGF-2 on isolated cardiac mitochondria is unknown. Hypothesis (3). FGF-2 exerts a direct protective effects on SSM. Direct stimulation of SSM with FGF-2 increased pS262-and-S368-mitoCx43, and calcium resistance to mPTP, and was dependent on mitoPKCε. FGF receptor inhibitors, SU5402 and FGFR neutralizing antibodies, blocked the effect of FGF-2, suggesting a FGFR1-like protein is responsible for the direct protective effects of FGF-2. This new intracellular mechanism of cytoprotection implies that endogenous intracellular levels of FGF-2 may determine constitutive levels of cardiac mitochondrial resistance to mPTP.October 201

Odolnost myokardu k ischemicko/reperfuznímu poškození - možné ochranné mechanizmy

Ischemická choroba srdeční představuje hlavní příčinu mortality v České republice a v dalších ekonomicky vyspělých zemích světa. Účinky ischemické choroby srdeční bývají připisovány patologickým změnám vyvolaným její akutní formou, infarktem myokardu. Tato disertační práce se pokusila přispět k celosvětové snaze o hlubší porozumění mechanismů, které by dokázaly ochránit srdce před důsledky ischemicko/reperfuzního (I/R) poškození. Disertační práce je založena na čtyřech publikacích - zatímco první tři jsou přijaté do tisku, čtvrtá se nachází v recenzním řízení. V první studii jsme prokázali úlohu oxidu dusnatého (NO) v mechanismu účinku chronické hypoxie (CH). Zjistili jsme, že exogenně zvýšená dostupnost NO stejně jako inhibice fosfodiesterázy typu 5 vede ke zvýšené ischemické toleranci srdcí normoxických i chronicky hypoxických potkanů. Účinky obou zásahů nebyly aditivní, což napovídá o zapojení signalizace spojené s NO v kardioprotektivním účinku CH. Druhá studie pokračovala ve studiu molekulárních mechanismů spojených s kardioprotekcí vyvolanou CH. Ukázali jsme, že protektivní efekt CH na velikost infarktu je spojen se zvýšenou koncentrací tumor nekrotizujícího faktoru alfa (TNF-α) a TNF-α receptoru R2. Ve třetí studii jsme sledovali účinek dexrazoxanu (DEX), dosud jediného klinicky schváleného...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...Katedra fyziologieDepartment of PhysiologyFaculty of SciencePřírodovědecká fakult

Odolnost myokardu k ischemicko/reperfuznímu poškození - možné ochranné mechanizmy

Ischemická choroba srdeční představuje hlavní příčinu mortality v České republice a v dalších ekonomicky vyspělých zemích světa. Účinky ischemické choroby srdeční bývají připisovány patologickým změnám vyvolaným její akutní formou, infarktem myokardu. Tato disertační práce se pokusila přispět k celosvětové snaze o hlubší porozumění mechanismů, které by dokázaly ochránit srdce před důsledky ischemicko/reperfuzního (I/R) poškození. Disertační práce je založena na čtyřech publikacích - zatímco první tři jsou přijaté do tisku, čtvrtá se nachází v recenzním řízení. V první studii jsme prokázali úlohu oxidu dusnatého (NO) v mechanismu účinku chronické hypoxie (CH). Zjistili jsme, že exogenně zvýšená dostupnost NO stejně jako inhibice fosfodiesterázy typu 5 vede ke zvýšené ischemické toleranci srdcí normoxických i chronicky hypoxických potkanů. Účinky obou zásahů nebyly aditivní, což napovídá o zapojení signalizace spojené s NO v kardioprotektivním účinku CH. Druhá studie pokračovala ve studiu molekulárních mechanismů spojených s kardioprotekcí vyvolanou CH. Ukázali jsme, že protektivní efekt CH na velikost infarktu je spojen se zvýšenou koncentrací tumor nekrotizujícího faktoru alfa (TNF-α) a TNF-α receptoru R2. Ve třetí studii jsme sledovali účinek dexrazoxanu (DEX), dosud jediného klinicky schváleného...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...Department of PhysiologyKatedra fyziologieFaculty of SciencePřírodovědecká fakult

Epitranskriptomika a kardioprotektivní intervence

1 Ischemic heart disease stands as the foremost global cause of mortality. Myocardial ischemia results in damage to cardiomyocytes which can further lead to impaired heart function. However, the extent of ischemic injury hinges not only on the intensity and duration of the ischemic stimulus but also on cardiac tolerance to ischemia. Therefore, it is extremely important to unravel the molecular basis of cardioprotective interventions such as adaptation to chronic hypoxia or fasting. We focused on the novel epitranscriptomic mechanisms around RNA modifications - N6 -methyladenosine (m6 A) and N6 ,2'-O-dimethyladenosine (m6 Am). Our findings revealed that while most epitranscriptomic modifiers displayed differential regulation in the heart following hypoxic adaptation and fasting, demethylases (ALKBH5 and FTO) were consistently upregulated after these cardioprotective interventions. Furthermore, we detected a discernible reduction in cardiac total RNA methylation levels after fasting. On the contrary, transcripts Nox4 and Hdac1, both of which play a role in the cytoprotective action of ketone bodies, exhibited increased methylation in hearts of fasting rats. Finally, inhibition of epitranscriptomic demethylases ALKBH5 and FTO decreased the hypoxic tolerance of adult rat primary cardiomyocytes isolated from...1 Ischemická choroba srdeční je celosvětově nejčastější příčinou úmrtí. Ischemie myokardu vede k poškození kardiomyocytů, což může vést k poruše srdeční funkce. Rozsah ischemického poškození však závisí nejen na intenzitě a délce trvání ischemického podnětu, ale také na toleranci srdce vůči ischemii. Objasnění molekulárního pozadí kardioprotektivních intervencí, jako je adaptace na chronickou hypoxii nebo hladovění, tak nabývá zásadního významu. Proto jsme se zaměřili na nové epitranskriptomické regulace dvou rozšířených modifikací RNA - N6 -methyladenosinu (m6 A) a N6 ,2'- O-dimethyladenosinu (m6 Am). Zjistili jsme, že většina epitranskriptomických regulátorů v srdci reaguje na hypoxickou adaptaci a na hladovění odlišným způsobem, demetylázy (ALKBH5 a FTO) byly ale v obou případech zvýšeny. Po hladovění bylo navíc v srdci patrné znatelné snížení hladin celkové metylace RNA. Hladina metylace v transkriptech Nox4 a Hdac1, které se účastní cytoprotektivních drah spouštěných ketolátkami, ale byla naopak zvýšena. V neposlední řadě, inhibice epitranskriptomických demetyláz ALKBH5 a FTO vedla ke snížení hypoxické tolerance kardiomyocytů izolovaných z hladovějících potkanů. Celkově naše zjištění ukazují na regulaci epitranskriptomických modifikací m6 A a m6 Am při kardioprotektivních intervencích, jako je adaptace...Department of PhysiologyKatedra fyziologiePřírodovědecká fakultaFaculty of Scienc

The effect of ischemic preconditioning on neuromuscular fatigue

Ischemic preconditioning (IPC) was initially developed as a protective technique against ischemia/reperfusion injuries, however it has since been employed as an ergogenic aid. Existing literature has demonstrated that IPC can elicit small ergogenic effects, which have been attributed to improved skeletal muscle oxygen delivery and extraction. However, recent studies have also suggested that IPC may augment neural drive, evidenced by improved surface electromyography (sEMG) signal parameters recorded concomitant to improved exercise performance. However, inferences made from sEMG measures warrant further investigation with more accurate assessments (i.e. interpolated twitch technique) in order to substantiate notions of augmented neural drive. Therefore, this thesis investigated the capacity of IPC to augment neural drive via central and peripheral mechanisms associated with sustaining neuromuscular function during exercise. Study 1 attempted to define any changes in neural drive that IPC may elicit following a sustained maximal isometric contraction. Whilst IPC improved skeletal muscle oxygen delivery and extraction, central and peripheral fatigue development remained unaffected. The use of an isometric exercise protocol in Study 1 may have inhibited any neural effects by preventing adequate muscle perfusion. Therefore, Study 2 utilised a maximal dynamic exercise protocol to assess the effect of IPC on neural drive. The addition of a dynamic exercise protocol made no impact on the capacity of IPC to augment neural drive. Furthermore, no benefits to oxygen delivery or extraction were observed throughout the exercise, suggesting the humoral benefits conferred by IPC may be more related to the impairment of tissue oxygenation imposed by the exercise. The final study (Study 3) of this thesis combined both dynamic exercise and tissue oxygenation deprivation (via hypoxia) in an attempt to create optimal conditions for observing an effect of IPC on neural drive. The use of hypoxia successfully demonstrated a relationship between impaired tissue oxygenation and the humoral benefits of IPC, however as previously observed, the capacity for IPC to elicit any significant effect on neuromuscular function remained non-existent. This thesis suggests that IPC is unable to influence parameters of neural drive during and following maximal single limb isometric and dynamic exercise. However, the findings suggest a task-dependant relationship between the humoral benefits of IPC and the level of tissue deoxygenation imposed by the exercise. Overall, the cumulative body of work in this thesis provides novel insights into the relationship between IPC and neuromuscular mechanisms associated with attenuating exercise-induced skeletal muscle fatigue