Charakterisierung des Guaninnukleotid-Austauschfaktors p63RhoGEF als neuen Mediator der Galphaq/11-induzierten RhoA-Aktivierung

Monomere GTPasen der Rho-Familie spielen als zentrale Mediatoren eine entscheidende Rolle bei der Regulation multipler zellulärer Prozesse, wie der Reorganisation des Aktin-Zytoskeletts, der Genexpression, Proliferation, Migration und Apoptose. Bis heute ist jedoch nur wenig über die unmittelbar beteiligten akzessorischen Proteine und Signalübertragungswege bekannt. Ziel dieser Arbeit war es daher, die Funktion des neu identifizierten, Galphaq/11-spezifischen Guaninnukleotid-Austauschfaktors (GEF) p63RhoGEF näher zu charakterisieren, zu untersuchen ob er an der RhoA-Aktivierung in kardialen Primärzellen der Ratte beteiligt ist und welche Bedeutung er bei der Entwicklung einer zellulären Hypertrophie in isolierten Kardiomyozyten hat. Zunächst wurden die biochemischen Eigenschaften von p63RhoGEF im Vergleich zum Leukemia-associated RhoGEF (LARG), das ebenfalls als Gq/11-reguliert beschrieben war, analysiert. Für beide GEFs konnte die Aktivierung über Galphaq/11-Proteine bestätigt werden, wobei unterschiedliche Mechanismen dieser Aktivierung zu Grunde liegen. Während für die Aktivierung von LARG, zusäztlich zur Bereitstellung von aktiviertem Galphaq/11, ein direkter Kontakt des GEFs mit dem Agonist-gebunden Gq/11PCR benötigt wird, ist für die Stimulation von p63RhoGEF eine direkte Interaktion mit aktivierten Proteinen der Galphaq/11-Familie ausreichend. Wie durch Analyse der Kristallstruktur von p63RhoGEF und Punktmutationen gezeigt, umfasst dabei die Kernregion der Bindung den C-terminalen Bereich der PH-Domäne („PH-Extension“) und wird zusätzlich durch einzelne Aminosäuren innerhalb der DH- und der PH-Domäne stabilisiert. Vergleichbare Charakteristika zeigen auch die beiden p63RhoGEF-Homologe Kalirin/Duet und Trio, so dass die drei GEFs zu einer neuen Dbl-Unterfamilie zusammengefasst werden können, die sich entwicklungsbiologisch von einem schon in Invertebraten (z.B. Unc73 in C.elegans) vorhanden Vetreter dieser Familie ableiten. Die Stimulation isolierter Kardiomyozyten der neonatalen Ratte (NRCM) mit Phenylephrin (PE) und Endothelin-1 (ET-1) induzierte eine partiell über Galphaq/11-vermittelte Aktivierung von RhoA. Im Gegensatz zu PE, führte dabei die ET-1-induzierte RhoA-Aktivierung zur Stressfaserbildung und dem Aufbau sarkomerer Strukturen, wobei der Galphaq/11-abhängige Anteil über p63RhoGEF vermittelt wurde. Klassische Hypertrophie-Indikatoren (verstärkte Proteinsynthese, Expression von Markerproteinen) zeigten keine Abhängigkeit von p63RhoGEF bzw. RhoA, sondern wurden über den Galphaq/11-Effektor PLCß sowie die monomere GTPase Rac1 reguliert. Die Stimulation von isolierten kardialen Fibroblasten mit Angiotensin II induzierte eine RhoA-Aktivierung, die selektiv über den Angiotensin II–Rezeptoren-Typ I - Galphaq/11 - p63RhoGEF-Signalweg erfolgte. Die Daten belegen eine Galphaq/11-p63RhoGEF-vermittelte RhoA-Aktivierung sowohl in Kardiomyozyten als auch in kardialen Fibroblasten. Da Angiotensin II ein wichtiger Mediator der pathologischen Fibrose im Herzen ist, könnte dieser Signalweg von pathophysiologischer Bedeutung sein

Phosphodiesterase 2 Protects against Catecholamine-induced Arrhythmias and Preserves Contractile Function after Myocardial Infarction

International audienceRationale: Phosphodiesterase 2 is a dual substrate esterase, which has the unique property to be stimulated by cGMP, but primarily hydrolyzes cAMP. Myocardial phosphodiesterase 2 is upregulated in human heart failure, but its role in the heart is unknown.Objective: To explore the role of phosphodiesterase 2 in cardiac function, propensity to arrhythmia, and myocardial infarction.Methods and Results: Pharmacological inhibition of phosphodiesterase 2 (BAY 60–7550, BAY) led to a significant positive chronotropic effect on top of maximal β-adrenoceptor activation in healthy mice. Under pathological conditions induced by chronic catecholamine infusions, BAY reversed both the attenuated β-adrenoceptor–mediated inotropy and chronotropy. Conversely, ECG telemetry in heart-specific phosphodiesterase 2-transgenic (TG) mice showed a marked reduction in resting and in maximal heart rate, whereas cardiac output was completely preserved because of greater cardiac contraction. This well-tolerated phenotype persisted in elderly TG with no indications of cardiac pathology or premature death. During arrhythmia provocation induced by catecholamine injections, TG animals were resistant to triggered ventricular arrhythmias. Accordingly, Ca2+-spark analysis in isolated TG cardiomyocytes revealed remarkably reduced Ca2+ leakage and lower basal phosphorylation levels of Ca2+-cycling proteins including ryanodine receptor type 2. Moreover, TG demonstrated improved cardiac function after myocardial infarction.Conclusions: Endogenous phosphodiesterase 2 contributes to heart rate regulation. Greater phosphodiesterase 2 abundance protects against arrhythmias and improves contraction force after severe ischemic insult. Activating myocardial phosphodiesterase 2 may, thus, represent a novel intracellular antiadrenergic therapeutic strategy protecting the heart from arrhythmia and contractile dysfunction

CNP Promotes Antiarrhythmic Effects via Phosphodiesterase 2

Background: Ventricular arrhythmia and sudden cardiac death are the most common lethal complications after myocardial infarction. Antiarrhythmic pharmacotherapy remains a clinical challenge and novel concepts are highly desired. Here, we focus on the cardioprotective CNP (C-type natriuretic peptide) as a novel antiarrhythmic principle. We hypothesize that antiarrhythmic effects of CNP are mediated by PDE2 (phosphodiesterase 2), which has the unique property to be stimulated by cGMP to primarily hydrolyze cAMP. Thus, CNP might promote beneficial effects of PDE2-mediated negative crosstalk between cAMP and cGMP signaling pathways. Methods: To determine antiarrhythmic effects of cGMP-mediated PDE2 stimulation by CNP, we analyzed arrhythmic events and intracellular trigger mechanisms in mice in vivo, at organ level and in isolated cardiomyocytes as well as in human-induced pluripotent stem cell-derived cardiomyocytes. Results: In ex vivo perfused mouse hearts, CNP abrogated arrhythmia after ischemia/reperfusion injury. Upon high-dose catecholamine injections in mice, PDE2 inhibition prevented the antiarrhythmic effect of CNP. In mouse ventricular cardiomyocytes, CNP blunted the catecholamine-mediated increase in arrhythmogenic events as well as in ICaL, INaL, and Ca2+spark frequency. Mechanistically, this was driven by reduced cellular cAMP levels and decreased phosphorylation of Ca2+handling proteins. Key experiments were confirmed in human iPSC-derived cardiomyocytes. Accordingly, the protective CNP effects were reversed by either specific pharmacological PDE2 inhibition or cardiomyocyte-specific PDE2 deletion. Conclusions: CNP shows strong PDE2-dependent antiarrhythmic effects. Consequently, the CNP-PDE2 axis represents a novel and attractive target for future antiarrhythmic strategies

Atropine augments cardiac contractility by inhibiting cAMP-specific phosphodiesterase type 4

Abstract Atropine is a clinically relevant anticholinergic drug, which blocks inhibitory effects of the parasympathetic neurotransmitter acetylcholine on heart rate leading to tachycardia. However, many cardiac effects of atropine cannot be adequately explained solely by its antagonism at muscarinic receptors. In isolated mouse ventricular cardiomyocytes expressing a Förster resonance energy transfer (FRET)-based cAMP biosensor, we confirmed that atropine inhibited acetylcholine-induced decreases in cAMP. Unexpectedly, even in the absence of acetylcholine, after G-protein inactivation with pertussis toxin or in myocytes from M2- or M1/3-muscarinic receptor knockout mice, atropine increased cAMP levels that were pre-elevated with the β-adrenergic agonist isoproterenol. Using the FRET approach and in vitro phosphodiesterase (PDE) activity assays, we show that atropine acts as an allosteric PDE type 4 (PDE4) inhibitor. In human atrial myocardium and in both intact wildtype and M2 or M1/3-receptor knockout mouse Langendorff hearts, atropine led to increased contractility and heart rates, respectively. In vivo, the atropine-dependent prolongation of heart rate increase was blunted in PDE4D but not in wildtype or PDE4B knockout mice. We propose that inhibition of PDE4 by atropine accounts, at least in part, for the induction of tachycardia and the arrhythmogenic potency of this drug

Inflammation leads through PGE/EP3 signaling to HDAC5/MEF2‐dependent transcription in cardiac myocytes

The myocyte enhancer factor 2 (MEF2) regulates transcription in cardiac myocytes and adverse remodeling of adult hearts. Activators of G protein‐coupled receptors (GPCRs) have been reported to activate MEF2, but a comprehensive analysis of GPCR activators that regulate MEF2 has to our knowledge not been performed. Here, we tested several GPCR agonists regarding their ability to activate a MEF2 reporter in neonatal rat ventricular myocytes. The inflammatory mediator prostaglandin E2 (PGE2) strongly activated MEF2. Using pharmacological and protein‐based inhibitors, we demonstrated that PGE2 regulates MEF2 via the EP3 receptor, the βγ subunit of Gi/o protein and two concomitantly activated downstream pathways. The first consists of Tiam1, Rac1, and its effector p21‐activated kinase 2, the second of protein kinase D. Both pathways converge on and inactivate histone deacetylase 5 (HDAC5) and thereby de‐repress MEF2. In vivo, endotoxemia in MEF2‐reporter mice induced upregulation of PGE2 and MEF2 activation. Our findings provide an unexpected new link between inflammation and cardiac remodeling by de‐repression of MEF2 through HDAC5 inactivation, which has potential implications for new strategies to treat inflammatory cardiomyopathies