Abstract Intracellular signalling cascades regulate cardiomyocyte hypertrophic response. Initially hypertrophy of individual myocytes occurs as an adaptive response to increased demands for cardiac work, e.g. during hypertension or after myocardial infarction, but a prolonged hypertrophic response, accompanied by accelerated fibrosis and apoptosis, predisposes the heart to impaired performance and the syndrome of heart failure. The goal of this work was to elucidate some of the main signalling pathways in experimental models of the cardiac hypertrophic response. Mechanical stretching of cultured neonatal rat cardiomyocytes in vitro activates the B-type natriuretic peptide (BNP) gene, a well-established marker of the hypertrophic response, through intracellular signalling cascades mitogen-activated protein kinases (MAPKs) and protein kinase A (PKA) -pathway. Further, transcription factors transcriptional enhancer factor-1 (TEF-1) and activating transcription factor 3 (ATF3) were induced during stretch, and TEF-1 activation was shown to be regulated by extracellular signal-regulated kinase (ERK), while ATF3 activation was modulated by PKA. The BNP gene was also activated by the adenoviral overexpression of the p38 MAPK isoforms p38α and p38β in vitro. Importantly, p38α–induced activation was mediated through activator protein-1 (AP-1) while p38β mediated BNP transcription through GATA-4, which suggests distinct physiological roles for different p38 isoforms. This was further confirmed by quantitative PCR, which demonstrated pro-fibrotic role for the p38α isoform and a pro-hypertrophic role for the p38β isoform. Finally, adenoviral overexpression of ATF3 in vitro and in vivo resulted in activation of cardiac survival factors nuclear factor-κВ and Nkx-2.5, and attenuation of central pro-inflammatory and pro-fibrotic mediators. Together these data suggest a protective role for ATF3 in the heart. Overall this study provides new insights into the role of several signalling molecules involved in cardiac hypertrophic process and suggests potential therapeutic strategies for the diagnosis and treatment of heart failure. / Tiivistelmä Sydämen kammioiden seinämät paksuuntuvat kuormituksen lisääntyessä mm. verenpainetaudissa tai sydäninfarktin jälkeen. Lisääntynyt kuormitus aiheuttaa sydänlihassolujen koon kasvun (hypertrofioitumisen) ohella sidekudoksen kertymistä (fibroosia) ja solukuolemaa. Nämä solutason muutokset lopulta vioittavat sydämen rakennetta niin, että sen toiminta pettää, ja sydän ajautuu vajaatoimintaan. Tätä taudin etenemistä säätelevät molekyylitasolla lukuisat solunsisäiset signaalinvälitysjärjestelmät, joita tässä väitöskirjatyössä tutkittiin eri koemalleissa. Sydämen täyttöpaineen nousun aiheuttama sydänlihassolujen mekaaninen venytys aktivoi natriureettisten peptidien (eteispeptidi, ANP ja B-tyypin natriureettinen peptidi, BNP) synteesiä ja vapautumista verenkiertoon. BNP geenin säätelyä mekaanisen venytyksen aikana tutkittiin rotan sydänlihassoluviljelmissä. Mitogeeni-aktivoituvat proteiinikinaasit (MAPK) sekä proteiinikinaasi A (PKA) säätelivät mekaanisen ärsykkeen aiheuttamaa BNP geenin ekspressiota. Venytys aktivoi myös transkriptiotekijöitä TEF-1 (transcriptional enhancer factor-1) ja ATF3 (activating transcription factor 3). TEF-1 sääteli venytyksen aiheuttamaa BNP:n aktivaatiota ERK:n (extracellular signal-regulated kinase) välityksellä BNP geenin säätelyalueella olevan sitoutumispaikkansa (M-CAT elementti) kautta. ATF3:n säätelyssä PKA:lla oli keskeinen merkitys. Tutkimus osoitti myös, että p38 MAPK:n alatyypeistä p38α lisäsi fibroosiin liittyvien geenien aktiivisuutta, kun taas p38β aiheutti solujen hypertrofioitumista lisäävien geenien ekspressiota. Molemmat alatyypit aktivoivat BNP geenin ekspressiota, mutta aktivaatio tapahtui eri transkriptiotekijöiden kautta. Tutkimuksessa havaittiin myös, että ATF3:n yliekspressio adenovirusvälitteisellä geeninsiirrolla lisäsi kahden sydäntä suojaavan transkriptiotekijän (nuclear factor-κВ ja Nkx-2.5) aktiivisuutta, sekä vähensi sydämen tulehdusvastetta ja fibroosia lisäävien tekijöiden (interleukiini-6 ja plasminogeeniaktivaattorin inhibiittori-1) ekspressiota. Väitöskirjatutkimus antaa uutta tietoa solunsisäisistä signaalinvälitys-järjestelmistä, jotka säätelevät sydänlihaksen kuormitusvastetta sydän- ja verenkiertoelimistön sairauksissa. Näiden solutason mekanismien tunteminen osaltaan edesauttaa jatkossa uusien menetelmien kehittämistä sydämen vajaatoiminnan ehkäisyyn ja hoitoon.

O treinamento por natação leva a uma sobrecarga de volume no coração, que induz a hipertrofia cardíaca (HC) excêntrica, com aumento da massa e do diâmetro cardíaco. Neste trabalho foram investigadas as adaptações no sistema cardiovascular e na expressão de genes relacionados à HC patológica, na gênese da HC por treinamento de natação. 42 ratas wistar foram divididas em grupos: sedentário controle (SC) treinado protocolo 1 (P1) e treinado protocolo 2 (P2). O treinamento de P1 foi de 1x60min/dia, 5x/semana, por 10 semanas. O de P2 foi igual ao P1 até a 8ª semana. Na 9ª semana 2x/dia e na 10ª semana 3x/dia. Os grupos treinados, em relação ao SC, apresentaram bradicardia de repouso, melhora no desempenho físico do teste máximo e do consumo máximo de oxigênio e HC, sem alterar a pressão arterial média e a expressão dos genes do fator natriurético atrial e da alfa actina esquelética. O grupo P2 apresentou aumento no diâmetro cardíaco e redução da expressão do gene da beta miosina de cadeia pesada. Este último resultado é contrário à literatura para a HC patológica, que mostra o aumento não só da expressão deste gene como a dos outros genes estudados. Os resultados de HC de P2 assemelham-se aos encontrados em estudos recentes com atletas de modalidades de maior componente aeróbio, sendo este um bom modelo para investigação dos mecanismos envolvidos na HC destes atleta / Swimming training leads to a cardiac volume overload that induces excentric cardiac hypertrophy (CH) with an increase in cardiac mass and diameter. Cardiovascular system adaptations and expression of genes relatated with pathological CH were investigated in swimming training CH. We studied 42 wistar females, divided in sedentary control (SC) group, protocol 1 trained group (P1) and protocol 2 trained group (P2). The P1 training program was once a day for 5 times/week for 10 weeks. P2 was the same as P1 until 8th week. In 9th week it was twice a day and in 10th week 3 times a day. Trained groups, in contrast with SC, showed rest bradycardia, improvement in physical performance, maximum oxygen uptake and CH, with no alteration in the medium arterial pressure and in the expression of atrial natriuretic factor and skeletal alpha actin genes. Moreover, P2 showed an increase in cardiac diameter and decrease in the expression of beta myosin heavy chain gene. This expression result is different of patological CH literature wich shows an increase of this gene expression and also in the others genes we had investigated. P2 CH results were similar to those recently found in endurance-type athletes, sugesting this is a good model to investigate mechanisms involved in endurance-type athletes CH

<p dir="ltr">Membrane-dependent enzymes play crucial roles in cellular signaling by transducing extracellular signals into intracellular responses. Phospholipase Cepsilon (PLCe) and diacylglycerol kinase alpha (DGKa) are membrane-associated enzymes regulated by G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), controlling signaling pathways essential for numerous cellular processes. PLCe catalyzes the hydrolysis of phosphatidylinositol phosphates into inositol phosphates (IPX) and diacylglycerol (DAG), triggering calcium release from intracellular stores and activating protein kinase C (PKC)-dependent pathways. While PLCe is crucial for normal cardiovascular function, hyperactivation or sustained activation can lead to hypertrophy. Due to structural heterogeneity, previous studies focused on isolated regulatory domains or the catalytic core. In this work, I present the first cryo-EM reconstruction of the largest PLCe fragment to date in complex with an antigen-binding fragment (Fab). This structure reveals the domain architecture of the N-terminal regions of the lipase and defines an extended membrane-binding surface critical for maximal basal and G protein-dependent activity. These findings lay the groundwork for high-resolution structures of the full-length enzyme and its complexes with the small GTPase Rap1A. Additionally, I explored the role of mAKAP in the Rap1A–PLCe pathway, alongside the guanine nucleotide exchange factor (GEF) function of PLCe toward Rap1A. In parallel, cryo-EM studies of DGKa bound to a covalent inhibitor were initiated. DGKa reduces DAG, thereby limiting PKC activity, and its inhibition is emerging as a promising cancer immunotherapy target. We have established a protocol for structural studies of full-length DGKa, which will elucidate its structures in basal and inhibited states.</p>

Die chronisch ischämische Herzkrankheit und der Myokardinfarkt (MI) sind die häufigsten Gründe für schwere Krankheit und vorzeitigen Tod in den entwickelten Ländern. Langfristig kommt es als Folge des Infarktes zur Kollateralgefäßbildung und zur Entwicklung einer kompensatorischen Herzhypertrophie. Eine Vielzahl von adaptativen Veränderungen in diesem Prozess konnte identifiziert werden. Wir konnten zeigen, dass in der akuten Phase nach MI Kontraktions- und Relaxationsgeschwindigkeit des Myokards erhöht waren. Die Expression der Hitzeschockproteine (HSP) 25 und 72 war verstärkt und korrelierte mit der Relaxationsgeschwindigkeit. In der chronischen Phase nach MI entwickelte sich eine signifikante Herzhypertrophie, die mit verminderter Kontraktions- und Relaxationsgeschwindigkeit einherging. Für die verlangsamte Relaxation war die verminderte Aktivität der Ca2+-ATPase des sarkoplasmatischen Retikulums (SERCA) als entscheidender Faktor anzusehen. Bei transgener Überexpression von Renin / Angiotensinogen ist die Relaxationsgeschwindigkeit des Myokards war wie auch nach MI durch geringere SERCA- Protein Expression vermindert. Die Empfindlichkeit der kontraktilen Funktion gegenüber Sauerstoffmangel und Reoxygenierung war nach MI gegenüber dem Kontrollmyokard geringer. Dafür konnten die verstärkte Expression der antioxidativ wirksamen HSPs und die erhöhte Aktivität der Glutathionperoxidase und der Superoxiddismutase, eine Verschiebung des Kreatinkinase (CK)- Isoenzymmusters und eine verminderte SERCA- Aktivität verantwortlich gemacht werden. Die Repolarisation der Aktionspotentiale der Kardiomyozyten war nach MI gegenüber den Kontrolltieren signifikant verlangsamt. Bereits eine 10-fach geringere artifizielle Dehnung des Gewebes führte nach MI im Vergleich zu Kontrolltieren zum Auftreten von Nachdepolarisationen und Extra-Aktionspotentialen. Ausschließlich in MI ließ sich durch die artifizielle Dehnung Vorhofflimmern auslösen, d.h. nach Myokardinfarkt war der mechano- elektrische Feedback Mechanismus empfindlicher. Die dehnungsinduzierten Veränderungen konnten durch Gadolinium unterdrückt werden, was auf eine Beteiligung von dehnungsaktivierten Ionenkanälen an den beobachteten Phänomenen schließen ließ. Auch kardiale Fibroblasten zeigten nach MI signifikante Änderungen ihrer elektrophysiologischen Eigenschaften, was zur Arrhythmieentstehung beitragen kann. Mittels molekularer Analysen konnten wir zeigen, dass der unter Sauerstoffmangel stabilisierte Transkriptionsfaktor Hif-1alpha in der Lage ist, den Promoter des Wilms' Tumor Suppressor Gens 1 (WT1) direkt transkriptionell zu aktivieren. Das führte zu verstärkter Expression von WT1 in den Herzen nach Myokardinfarkt, und zu verstärkter Expression von WT1 in Herz und Niere bei systemischer normobarer Hypoxie. Die WT1 Expression im Herzen nach MI ließ sich in den Koronargefäßen lokalisieren. Koexpression mit Proliferations- und Vaskulogenesemarkern ließ vermuten, dass WT1 nach MI eine wichtige Rolle für die Neovaskulogenese spielt. Die gewonnenen Ergebnisse tragen zum Verständnis der pathophysiologischen Veränderungen bei kardialer Hypertrophie nach Myokardinfarkt bei und eröffnen möglicherweise langfristig neue therapeutische Ansätze. / Chronic ischemic heart disease and myocardial infarction are the most common causes for morbidity and mortality in industrialized countries. A survived myocardial infarction (MI) results in a long run in collateral formation and the development of cardiac hypertrophy. A variety of adaptive responses in this process had been identified. We could show that in the acute phase after Mi in rats, contraction- and relaxation rates of the myocardium are increased. The higher relaxation rate correlates to an increased expression of heat shock proteins. In the chronic phase after MI, with the development of cardiac hypertrophy, contraction and relaxation rates decrease. The decrease in the relaxation rate could be attributed to a reduced activity of the Ca- ATPase of the sarcoplasmic reticulum (SERCA2). Transgenic overexpression of renin / angiotensinogen also resulted in a reduced SERCA2 expression and, consequently, lower relaxation rate. The susceptibility of contractile function to hypoxia - reoxygenation was reduced after MI compared to sham operated control animals. The lower susceptibility to hypoxia - reoxygenation could be attributed to an increased expression of heat shock proteins, higher activities of the antioxidant enzymes glutathionperoxidase and superoxiddismutase, shifts in the isoenzyme distribution of the creatine kinase, and a reduced SERCA2 activity. Repolarization of cardiomyocyte action potentials was found to be delayed after MI. A 10-fold lower artificial stretch of the tissue after MI than after sham operation caused afterdepolarizations and extra action potentials. Higher artificial stretch caused atrial fibrillation only after MI suggesting an intensified mechano-electrical feedback mechanism after MI. Stretch- induced electrical abnormalities could be suppressed by gadolinium suggesting the involvement of stretch-activated ion channels in the electrical abnormalities. Also electrophysiological properties of cardiac fibroblasts were significantly altered after MI, which may contribute to the increased risk for arrhythmia after infarction. Furthermore, we could show that the Hif-1alpha transcription factor, which is stabilized under hypoxic conditions is capable to directly activate the Wilms'' tumor suppressor 1 (WT1) transcriptionally. This leads to an increased expression of WT1 in the heart after MI and in heart and kidneys after systemic hypoxia. After MI, WT1 is expressed mainly in coronary vessels. Co-expression of WT1 with markers of proliferation and vasculogenesis suggests a role of WT1 in neovasculogenesis. These findings contribute to our understanding of pathophysiological alterations in the development of cardiac hypertrophy after MI and may contribute to the development of new therapeutic approaches.

Herzinsuffizienz mit konsekutivem Herzversagen ist ein zentrales kardiovaskuläres Problem der heutigen Bevölkerung.Ursächlich ist insbesondere eine progrediente Herzhypertrophie. Die Calcium-Calmodulin abhängige Phosphatase Calcineurin (CnR) spielt hierbei in der Pathogenese eine entscheidende Rolle. CnR wird über seine Calciumbindungsstellen an der regulatorischen Untereinheit Calcineurin B (CnB) aktiviert.Um zu untersuchen, inwieweit CnB bei der Hypertrophie verschiedener Ätiologien reguliert wird, wurde in linksventrikulären Myokardbiopsien von Patienten mit Aortenstenose (AS= 14) bzw. aus explantierten Herzen mit Dilatativer Kardiomyopathie (DCM=27) und Koronarer Herzerkrankung (KHK=7) der mRNA-und Proteingehalt von CnB bestimmt und mit der Expression von ANP und BNP korreliert. Als Kontrollgruppe dienten 15 abgelehnte Spenderherzen mit normaler systolischer Funktion und gesunder Morphologie. In den Herzen der Kontroll-, DCM-, und KHK-Gruppen wurde der linksventrikuläre Fibrosegehalt bestimmt. Hierzu wurden eine extern standardisierte Real-Time-PCR-Technik und ein etabliertes Western Blot Verfahren angewandt. Die Ergebnisse werden im Median ± 25%/75%-Perzentile angegeben und mit dem Mann-Whitney-Test bzw. Korrelationsanalysen nach Spearman berechnet. In den Herzen mit DCM zeigte sich eine signifikante Erhöhung der CnB mRNA auf ca. das Dreifache der Kontrollen (293% der Ko, p / Heart failure is a central cardiovascular problem for the current population. Cardiac hypertrophy is a central factor. The calcium-Calmodulin dependent phosphatase Calcineurin (CnR) plays a crucial role in the pathogenesis. CnR is activated via its calcium-binding site in the regulatory subunit Calcineurin B (CnB). In order to examine, to what extent CnB is regulated in different aetiologies of hypertrophy, we analysed CnB´s mRNA and protein in left ventricular samples from patients with aortic valve stenosis (AS = 14) and from explanted hearts with dilated (DCM=27) and ischemic (ICM=7) cardiomyopathy and correlated them with the expression of ANP and BNP. As a control, 15 rejected donor hearts with normal systolic function and non-pathologic changed morphology were used. Fibrosis of the left ventricle was determined in three groups: control , DCM and ICM. Therefore, we used an externally standardized real-time PCR and an established Western Blot. Data are given as median ± 25%/75%- percentiles; Mann Whitney test and Spearman´s correlation-analyses were used. CnB mRNA was significantly raised in DCM (293% of control, p

L’infarctus du myocarde (IM) survenant après l’obstruction de l’artère coronaire est la cause principale des décès cardiovasculaires. Après l’IM, le coeur endommagé répond à l’augmentation du stress hémodynamique avec une cicatrice et une hypertrophie dans la région non-infarcie du myocarde. Dans la région infarcie, la cicatrice se forme grâce au dépôt du collagène. Pendant formation de la cicatrice, les cardiomyocytes ventriculaires résidant dans la région non-infarcie subissent une réponse hypertrophique après l’activation chronique due au système sympathique et à l’angiotensine II. La cicatrisation préserve l’intégrité structurale du coeur et l'hypertrophie des cardiomyocytes apporte un support ionotropique. Le canal CaV1.2 joue un rôle dans la réponse hypertrophique après l’IM. L’activation du CaV1.2 déclenche la signalisation dépendante de Ca2+ induisant l’hypertrophie. Cependant, il est rapporté que l’ouverture des canaux potassiques (KATP) ATP sensitifs joue un rôle sélectif dans l’expansion de la cicatrice après IM. Malgré leur expression dans les coeurs mâles, les KATP fournissent une cardioprotection sexe dépendante limitant l’expansion de la cicatrice chez les femelles. L’administration de rapamycine aux rates ayant subi un infarctus produit l’expansion de la cicatrice, soutenant la relation possible entre la cible de rapamycine, mTORC1 et les KATP dans la cardioprotection sexe spécifique. Effectivement, dans les cellules pancréatiques α, la signalisation mTORC1 était couplée à l'activation du KATP. Cependant, le lien entre mTORC1 et les canaux KATP dans le coeur reste inconnu. L'objectif de la thèse est d’examiner le rôle des canaux ioniques dans le remodelage cardiaque post-IM, surtout des canaux calciques dans l'hypertrophie et d'élucider la relation entre les KATP et mTORC1. L’hypothèse première teste que l’hypertrophie médiée par le système sympathique des cardiomyocytes ventriculaires des rats néonataux (NRCM) produit une augmentation de l’influx calcique après une augmentation des sous-unités du CaV1.2. Le traitement de norépinéphrine (NE) quadruple l’amplitude du courant calcique type L et double l’expression protéique des sous unités de CaVα2δ1 et CaVβ3. L’hypertrophie des NRCM au NE s’associe à une augmentation de la phosphorylation de la Kinase ERK 1/2. Le β1-bloqueur metoprolol et l’inhibiteur ii de ERK1/2 diminuent l’effet de NE sur CaVα2δ1. Cependant, l’augmentation de CaVβ3 et de la réponse hypertrophique persiste. Ainsi, le signal β1-adrenergique à travers ERK augmente les sous-unités CaVα2δ1 outre l’hypertrophie. L’autre hypothèse examine la spécificité du sexe sur l’expansion cicatricielle médiée par rapamycine et l’influence de mTOR sur l’expression de KATP. Rapamycin augmente la surface de la cicatrice et inhibe la phosphorylation de mTOR chez les coeurs de femelles. Dans les coeurs des deux sexes, la phosphorylation de mTOR et l’expression de KATP, Kir6.2 et SUR2A sont similaires. Cependant, une grande inactivation de la tubérine et une faible expression de raptor sont détectées chez les femelles. Le traitement à l’ester de phorbol des NRCM induit l’hypertrophie, augmente la phosphorylation de p70S6K et l’expression SUR2A. Le prétraitement par Rapamycine atténue chacune des réponses. Rapamycin démontre un patron d’expansion cicatriciel sexe spécifique et une régulation de phosphorylation de mTOR dans IM. Aussi, l’augmentation de SUR2A dans les NRCM traités par PDBu révèle une interaction entre mTOR et KATP. / Myocardial infarction (MI) secondary to the obstruction of the coronary artery is the main cause of cardiovascular death. Following MI, the damaged heart adapts to the increased hemodynamic stress via formation of a scar and a hypertrophic response of ventricular cardiomyocytes in the non-infarcted myocardium. In the infarcted region, a scar is formed via the rapid deposition of collagen. With ongoing scar formation, ventricular cardiomyocytes in the non-infarcted myocardium undergo a hypertrophic response secondary to the chronic activation by the sympathetic system and angiotensin II. Collectively, scar formation and cardiomyocyte hypertrophy preserve the structural integrity of the heart and provide inotropic support, respectively. CaV1.2 channels play a significant role in the hypertrophic response post-MI. Notably, the activation of CaV1.2 channel triggers Ca2+-dependent signaling that induces hypertrophy. By contrast, the opening of ATP-sensitive potassium (KATP) channels was shown to partake in selective scar expansion following MI. Notwithstanding its expression in male hearts, KATP channels endow a sex-dependent cardioprotection limiting scar expansion selectively in females. Moreover, administration of the macrolide rapamycin to the infarcted female rat heart led to scar expansion, supporting the possible relationship between the target of rapamycin, mTORC1 and KATP channels in providing sex-specific cardioprotection. Indeed, in pancreatic-α cells, mTORC1 signaling was coupled to KATP channel activation. However, whether mTORC1 targets KATP channels in the heart remains unknown. Thus, the AIM of the thesis was to explore the role of ion channels in cardiac remodeling post-MI by specifically addressing the role of Ca channels in cardiomyocyte hypertrophy and elucidate the potential relationship between KATP channels and mTORC1 signaling. The first study tested the hypothesis that hypertrophied neonatal rat ventricular cardiomyocytes (NRVMs) following sympathetic stimulation translated to an increase in calcium influx secondary to the augmentation of CaV1.2 channel subunits. NE treatment led to a 4-fold increase of L-type Ca2+ peak current associated with a 2-fold upregulation of CaVα2δ1 and CaVβ3 protein subunits in hypertrophied NRVMs. The hypertrophic response of NNVMs to NE was associated with the increased phosphorylation of extracellular regulated kinase (ERK1/2). The β1-blocker metoprolol and the ERK1/2 inhibitor suppressed NE-mediated protein upregulation of CaVα2δ1 whereas CaVβ3 upregulation and the hypertrophic response persisted. Therefore, sympathetic mediated β1-adrenergic signaling via ERK selectively upregulated the CaVα2δ1 subunit independent of NRVM hypertrophy. The second study tested the hypothesis that rapamycin-mediated scar expansion was sexspecific and mTOR influenced KATP channel subunit expression. Rapamycin administration translated to scar expansion and inhibited mTOR phosphorylation exclusively in females. In normal adult male and female rat hearts, mTOR phosphorylation and protein levels of KATP channel subunits Kir6.2 and SUR2A were similar. However, greater tuberin inactivation and reduced raptor protein levels were detected in females. NRVMs treated with a phorbol ester induced hypertrophy, increased p70S6K phosphorylation and SUR2A protein levels and rapamycin pretreatment attenuated each response. Thus, rapamycin administration to MI rats unmasked a sex-specific pattern of scar expansion and highlighted the disparate regulation of mTOR phosphorylation. Moreover, rapamycin-dependent upregulation of SUR2A in PDButreated NRVMs revealed a novel interaction between mTOR and KATP channel subunit expression