Matthias Dewenter

Phosphodiesterase-2 Is Up-Regulated in Human Failing Hearts and Blunts β-Adrenergic Responses in Cardiomyocytes

OBJECTIVES This study investigated whether myocardial phosphodiesterase-2 (PDE2) is altered in heart failure (HF) and determined PDE2-mediated effects on beta-adrenergic receptor (β-AR) signaling in healthy and diseased cardiomyocytes. BACKGROUND Diminished cyclic adenosine monophosphate (cAMP) and augmented cyclic guanosine monophosphate (cGMP) signaling is characteristic for failing hearts. Among the PDE superfamily, PDE2 has the unique property of being able to be stimulated by cGMP, thus leading to a remarkable increase in cAMP hydrolysis mediating a negative cross talk between cGMP and cAMP signaling. However, the role of PDE2 in HF is poorly understood. METHODS Immunoblotting, radioenzymatic- and fluorescence resonance energy transfer-based assays, video edge detection, epifluorescence microscopy, and L-type Ca2(+) current measurements were performed in myocardial tissues and/or isolated cardiomyocytes from human and/or experimental HF, respectively. RESULTS Myocardial PDE2 expression and activity were ~2-fold higher in advanced human HF. Chronic β-AR stimulation via catecholamine infusions in rats enhanced PDE2 expression ~2-fold and cAMP hydrolytic activity ~4-fold, which correlated with blunted cardiac β-AR responsiveness. In diseased cardiomyocytes, higher PDE2 activity could be further enhanced by stimulation of cGMP synthesis via nitric oxide donors, whereas specific PDE2 inhibition partially restored β-AR responsiveness. Accordingly, PDE2 overexpression in healthy cardiomyocytes reduced the rise in cAMP levels and L-type Ca2(+) current amplitude, and abolished the inotropic effect following acute β-AR stimulation, without affecting basal contractility. Importantly, PDE2-overexpressing cardiomyocytes showed marked protection from norepinephrine-induced hypertrophic responses. CONCLUSIONS PDE2 is markedly up-regulated in failing hearts and desensitizes against acute β-AR stimulation. This may constitute an important defense mechanism during cardiac stress, for example, by antagonizing excessive β-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular antiadrenergic therapeutic strategy in HF.

Function and regulation of serine/threonine phosphatases in the healthy and diseased heart

Protein phosphorylation is a major control mechanism of a wide range of physiological processes and plays an important role in cardiac pathophysiology. Serine/threonine protein phosphatases control the dephosphorylation of a variety of cardiac proteins, thereby fine-tuning cardiac electrophysiology and function. Specificity of protein phosphatases type-1 and type-2A is achieved by multiprotein complexes that target the catalytic subunits to specific subcellular domains. Here, we describe the composition, regulation and target substrates of serine/threonine phosphatases in the heart. In addition, we provide an overview of pharmacological tools and genetic models to study the role of cardiac phosphatases. Finally, we review the role of protein phosphatases in the diseased heart, particularly in ventricular arrhythmias and atrial fibrillation and discuss their role as potential therapeutic targets.

PDE2-mediated cAMP hydrolysis accelerates cardiac fibroblast to myofibroblast conversion and is antagonized by exogenous activation of cGMP signaling pathways

Recent studies suggest that the signal molecules cAMP and cGMP have antifibrotic effects by negatively regulating pathways associated with fibroblast to myofibroblast (MyoCF) conversion. The phosphodiesterase 2 (PDE2) has the unique property to be stimulated by cGMP, which leads to a remarkable increase in cAMP hydrolysis and thus mediates a negative cross-talk between both pathways. PDE2 has been recently investigated in cardiomyocytes; here we specifically addressed its role in fibroblast conversion and cardiac fibrosis. PDE2 is abundantly expressed in both neonatal rat cardiac fibroblasts (CFs) and cardiomyocytes. The overexpression of PDE2 in CFs strongly reduced basal and isoprenaline-induced cAMP synthesis, and this decrease was sufficient to induce MyoCF conversion even in the absence of exogenous profibrotic stimuli. Functional stress-strain experiments with fibroblast-derived engineered connective tissue (ECT) demonstrated higher stiffness in ECTs overexpressing PDE2. In regard to cGMP, neither basal nor atrial natriuretic peptide-induced cGMP levels were affected by PDE2, whereas the response to nitric oxide donor sodium nitroprusside was slightly but significantly reduced. Interestingly, despite persistently depressed cAMP levels, both cGMP-elevating stimuli were able to completely prevent the PDE2-induced MyoCF phenotype, arguing for a double-tracked mechanism. In conclusion, PDE2 accelerates CF to MyoCF conversion, which leads to greater stiffness in ECTs. Atrial natriuretic peptide- and sodium nitroprusside-mediated cGMP synthesis completely reverses PDE2-induced fibroblast conversion. Thus PDE2 may augment cardiac remodeling, but this effect can also be overcome by enhanced cGMP. The redundant role of cAMP and cGMP as antifibrotic meditators may be viewed as a protective mechanism in heart failure.

Phosphodiesterase 2 Protects Against Catecholamine-Induced Arrhythmia and Preserves Contractile Function After Myocardial Infarction

Rationale: 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 &bgr;-adrenoceptor activation in healthy mice. Under pathological conditions induced by chronic catecholamine infusions, BAY reversed both the attenuated &bgr;-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.

Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

AIMS The cAMP-dependent protein kinase (PKA) mediates β-adrenoceptor (β-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. METHODS AND RESULTS Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. β-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. CONCLUSION Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to β-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to β-AR stimulation.

Calcium Signaling and Transcriptional Regulation in Cardiomyocytes

Calcium (Ca2+) is a universal regulator of various cellular functions. In cardiomyocytes, Ca2+ is the central element of excitation-contraction coupling, but also impacts diverse signaling cascades and influences the regulation of gene expression, referred to as excitation-transcription coupling. Disturbances in cellular Ca2+-handling and alterations in Ca2+-dependent gene expression patterns are pivotal characteristics of failing cardiomyocytes, with several excitation-transcription coupling pathways shown to be critically involved in structural and functional remodeling processes. Thus, targeting Ca2+-dependent transcriptional pathways might offer broad therapeutic potential. In this article, we (1) review cytosolic and nuclear Ca2+ dynamics in cardiomyocytes with respect to their impact on Ca2+-dependent signaling, (2) give an overview on Ca2+-dependent transcriptional pathways in cardiomyocytes, and (3) discuss implications of excitation-transcription coupling in the diseased heart.

A proteolytic fragment of histone deacetylase 4 protects the heart from failure by regulating the hexosamine biosynthetic pathway

The stress-responsive epigenetic repressor histone deacetylase 4 (HDAC4) regulates cardiac gene expression. Here we show that the levels of an N-terminal proteolytically derived fragment of HDAC4, termed HDAC4-NT, are lower in failing mouse hearts than in healthy control hearts. Virus-mediated transfer of the portion of the Hdac4 gene encoding HDAC4-NT into the mouse myocardium protected the heart from remodeling and failure; this was associated with decreased expression of Nr4a1, which encodes a nuclear orphan receptor, and decreased NR4A1-dependent activation of the hexosamine biosynthetic pathway (HBP). Conversely, exercise enhanced HDAC4-NT levels, and mice with a cardiomyocyte-specific deletion of Hdac4 show reduced exercise capacity, which was characterized by cardiac fatigue and increased expression of Nr4a1. Mechanistically, we found that NR4A1 negatively regulated contractile function in a manner that depended on the HBP and the calcium sensor STIM1. Our work describes a new regulatory axis in which epigenetic regulation of a metabolic pathway affects calcium handling. Activation of this axis during intermittent physiological stress promotes cardiac function, whereas its impairment in sustained pathological cardiac stress leads to heart failure.

Calcium/Calmodulin-Dependent Protein Kinase II Activity Persists During Chronic β-Adrenoceptor Blockade in Experimental and Human Heart Failure

Background— Considerable evidence suggests that calcium/calmodulin-dependent protein kinase II (CaMKII) overactivity plays a crucial role in the pathophysiology of heart failure (HF), a condition characterized by excessive &bgr;-adrenoceptor (&bgr;-AR) stimulation. Recent studies indicate a significant cross talk between &bgr;-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental &bgr;-AR signaling in HF. In this study, we investigated the effect of chronic &bgr;-AR blocker treatment on CaMKII activity in human and experimental HF. Methods and Results— Immunoblot analysis of myocardium from end-stage HF patients (n=12) and non-HF subjects undergoing cardiac surgery (n=12) treated with &bgr;-AR blockers revealed no difference in CaMKII activity when compared with non–&bgr;-AR blocker–treated patients. CaMKII activity was judged by analysis of CaMKII expression, autophosphorylation, and oxidation and by investigating the phosphorylation status of CaMKII downstream targets. To further evaluate these findings, CaMKII&dgr;C transgenic mice were treated with the &bgr;1-AR blocker metoprolol (270 mg/kg*d). Metoprolol significantly reduced transgene-associated mortality (n≥29; P<0.001), attenuated the development of cardiac hypertrophy (−14±6% heart weight/tibia length; P<0.05), and strongly reduced ventricular arrhythmias (−70±22% premature ventricular contractions; P<0.05). On a molecular level, metoprolol expectedly decreased protein kinase A–dependent phospholamban and ryanodine receptor 2 phosphorylation (−42±9% for P-phospholamban-S16 and −22±7% for P-ryanodine receptor 2-S2808; P<0.05). However, this was paralled neither by a reduction in CaMKII autophosphorylation, oxidation, and substrate binding nor a change in the phosphorylation of CaMKII downstream target proteins (n≥11). The lack of CaMKII modulation by &bgr;-AR blocker treatment was confirmed in healthy wild-type mice receiving metoprolol. Conclusions— Chronic &bgr;-AR blocker therapy in patients and in a mouse model of CaMKII-induced HF is not associated with a change in CaMKII activity. Thus, our data suggest that the molecular effects of &bgr;-AR blockers are not based on a modulation of CaMKII. Directly targeting CaMKII may, therefore, further improve HF therapy in addition to &bgr;-AR blockade.Background Considerable evidence suggests that CaMKII overactivity plays a crucial role in the pathophysiology of heart failure (HF), a condition characterized by excessive β-adrenoceptor (β-AR) stimulation. Recent studies indicate a significant crosstalk between β-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental β-AR signaling in HF. In this study we investigated the effect of chronic β-AR blocker treatment on CaMKII activity in human and experimental HF.

Chronic loss of inhibitor-1 diminishes cardiac RyR2 phosphorylation despite exaggerated CaMKII activity

Inhibitor-1 (I-1) modulates protein phosphatase 1 (PP1) activity and thereby counteracts the phosphorylation by kinases. I-1 is downregulated and deactivated in failing hearts, but whether its role is beneficial or detrimental remains controversial, and opposing therapeutic strategies have been proposed. Overactivity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) with hyperphosphorylation of ryanodine receptors (RyR2) at the CaMKII-site is recognized to be central for heart failure and arrhythmias. Using an I-1-deficient mouse line as well as transfected cell lines, we investigated the effects of acute and chronic modulation of I-1 on CaMKII activity and RyR2 phosphorylation. We demonstrate that I-1 acutely modulates CaMKII by regulating PP1 activity. However, while ablation of I-1 should thus limit CaMKII-activation, we unexpectedly found exaggerated CaMKII-activation under β-adrenergic stress upon chronic loss of I-1 in knockout mice. We unraveled that this is due to chronic upregulation of the exchange protein activated by cAMP (EPAC) leading to augmented CaMKII activation, and using computational modeling validated that an increase in EPAC expression can indeed explain our experimental findings. Interestingly, at the level of RyR2, the increase in PP1 activity more than outweighed the increase in CaMKII activity, resulting in reduced RyR phosphorylation at Ser-2814. Exaggerated CaMKII activation due to counterregulatory mechanisms upon loss of I-1 is an important caveat with respect to suggested therapeutic I-1-inhibition, as CaMKII overactivity has been heavily implicated in several cardiac pathologies.

LATITUDE-TIMI: is there still hope for anti-inflammatory therapy in acute myocardial infaction?

Inflammation is a crucial feature of atherosclerotic plaque formation as well as post myocardial infarction (MI) remodeling and thus thought to play an essential role in the pathogenesis of ischemic heart disease and its complications (1,2). The regenerative processes after MI involve inflammatory, proliferative and maturation phases that are crucial for wound healing and reparation (3). However, an overactive inflammatory and fibrotic response is associated with maladaptive structural and electrophysiological remodeling, leading to systolic as well as diastolic dysfunction and contributing to the development of an arrhythmogenic substrate (2,4). Moreover, elevated cytokine levels also correlate with increased risk for recurrent coronary events in patients with MI (5).