Regulation of heterotrimeric G-protein signaling by NDPK/NME proteins and caveolins: an update.

Heterotrimeric G proteins are pivotal mediators of cellular signal transduction in eukaryotic cells and abnormal G-protein signaling plays an important role in numerous diseases. During the last two decades it has become evident that the activation status of heterotrimeric G proteins is both highly localized and strongly regulated by a number of factors, including a receptor-independent activation pathway of heterotrimeric G proteins that does not involve the classical GDP/GTP exchange and relies on nucleoside diphosphate kinases (NDPKs). NDPKs are NTP/NDP transphosphorylases encoded by the nme/nm23 genes that are involved in a variety of cellular events such as proliferation, migration, and apoptosis. They therefore contribute, for example, to tumor metastasis, angiogenesis, retinopathy, and heart failure. Interestingly, NDPKs are translocated and/or upregulated in human heart failure. Here we describe recent advances in the current understanding of NDPK functions and how they have an impact on local regulation of G-protein signaling.

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

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 ß-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.

Calcium/Calmodulin-Dependent Protein Kinase II Activity Persists During Chronic ß-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 ß-adrenoceptor (ß-AR) stimulation. Recent studies indicate a significant cross talk between ß-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental ß-AR signaling in HF. In this study, we investigated the effect of chronic ß-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 ß-AR blockers revealed no difference in CaMKII activity when compared with non-ß-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, CaMKIIδC transgenic mice were treated with the ß1-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 ß-AR blocker treatment was confirmed in healthy wild-type mice receiving metoprolol. CONCLUSIONS: Chronic ß-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 ß-AR blockers are not based on a modulation of CaMKII. Directly targeting CaMKII may, therefore, further improve HF therapy in addition to ß-AR blockade.

Nucleoside Diphosphate Kinase-C Suppresses cAMP Formation in Human Heart Failure.

BACKGROUND: Chronic heart failure (HF) is associated with altered signal transduction via ß-adrenoceptors and G proteins and with reduced cAMP formation. Nucleoside diphosphate kinases (NDPKs) are enriched at the plasma membrane of patients with end-stage HF, but the functional consequences of this are largely unknown, particularly for NDPK-C. Here, we investigated the potential role of NDPK-C in cardiac cAMP formation and contractility. METHODS: Real-time polymerase chain reaction, (far) Western blot, immunoprecipitation, and immunocytochemistry were used to study the expression, interaction with G proteins, and localization of NDPKs. cAMP levels were determined with immunoassays or fluorescent resonance energy transfer, and contractility was determined in cardiomyocytes (cell shortening) and in vivo (fractional shortening). RESULTS: NDPK-C was essential for the formation of an NDPK-B/G protein complex. Protein and mRNA levels of NDPK-C were upregulated in end-stage human HF, in rats after long-term isoprenaline stimulation through osmotic minipumps, and after incubation of rat neonatal cardiomyocytes with isoprenaline. Isoprenaline also promoted translocation of NDPK-C to the plasma membrane. Overexpression of NDPK-C in cardiomyocytes increased cAMP levels and sensitized cardiomyocytes to isoprenaline-induced augmentation of contractility, whereas NDPK-C knockdown decreased cAMP levels. In vivo, depletion of NDPK-C in zebrafish embryos caused cardiac edema and ventricular dysfunction. NDPK-B knockout mice had unaltered NDPK-C expression but showed contractile dysfunction and exacerbated cardiac remodeling during long-term isoprenaline stimulation. In human end-stage HF, the complex formation between NDPK-C and Gαi2 was increased whereas the NDPK-C/Gαs interaction was decreased, producing a switch that may contribute to an NDPK-C-dependent cAMP reduction in HF. CONCLUSIONS: Our findings identify NDPK-C as an essential requirement for both the interaction between NDPK isoforms and between NDPK isoforms and G proteins. NDPK-C is a novel critical regulator of ß-adrenoceptor/cAMP signaling and cardiac contractility. By switching from Gαs to Gαi2 activation, NDPK-C may contribute to lower cAMP levels and the related contractile dysfunction in HF.

The activation of RhoC in vascular endothelial cells is required for the S1P receptor type 2-induced inhibition of angiogenesis.

Sphingosine-1-phosphate (S1P) is a multifunctional phospholipid inducing a variety of cellular responses in endothelial cells (EC). S1P responses are mediated by five G protein coupled receptors of which three types (S1P1R-S1P3R) have been described to be of importance in vascular endothelial cells (EC). Whereas the S1P1R regulates endothelial barrier function by coupling to Gαi and the monomeric GTPase Rac1, the signaling pathways involved in the S1P-induced regulation of angiogenesis are ill defined. We therefore studied the sprouting of human umbilical vein EC (HUVEC) in vitro and analyzed the activation of the RhoGTPases RhoA and RhoC. Physiological relevant concentrations of S1P (100-300nM) induce a moderate activation of RhoA and RhoC. Inhibition or siRNA-mediated depletion of the S1P2R preferentially decreased the activation of RhoC. Both manipulations caused an increase of sprouting in a spheroid based in vitro sprouting assay. Interestingly, a similar increase in sprouting was detected after effective siRNA-mediated knockdown of RhoC. In contrast, the depletion of RhoA had no influence on sprouting. Furthermore, suppression of the activity of G proteins of the Gα12/13 subfamily by adenoviral overexpression of the regulator of G protein signaling domain of LSC as well as siRNA-mediated knockdown of the Rho specific guanine nucleotide exchange factor leukemia associated RhoGEF (LARG) inhibited the S1P-induced activation of RhoC and concomitantly increased sprouting of HUVEC with similar efficacy. We conclude that the angiogenic sprouting of EC is suppressed via the S1P2R subtype. Thus, the increase in basal sprouting can be attributed to blocking of the inhibitory action of autocrine S1P stimulating the S1P2R. This inhibitory pathway involves the activation of RhoC via Gα12/13 and LARG, while the simultaneously occurring activation of RhoA is apparently dispensable here.

Phosphodiesterase-2 is up-regulated in human failing hearts and blunts ß-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 (ß-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 ß-AR stimulation via catecholamine infusions in rats enhanced PDE2 expression ~2-fold and cAMP hydrolytic activity ~4-fold, which correlated with blunted cardiac ß-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 ß-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 ß-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 ß-AR stimulation. This may constitute an important defense mechanism during cardiac stress, for example, by antagonizing excessive ß-AR drive. Thus, activating myocardial PDE2 may represent a novel intracellular antiadrenergic therapeutic strategy in HF.

Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling.

Angiopoietin-2 (ANG-2) is a key regulator of angiogenesis that exerts context-dependent effects on ECs. ANG-2 binds the endothelial-specific receptor tyrosine kinase 2 (TIE2) and acts as a negative regulator of ANG-1/TIE2 signaling during angiogenesis, thereby controlling the responsiveness of ECs to exogenous cytokines. Recent data from tumors indicate that under certain conditions ANG-2 can also promote angiogenesis. However, the molecular mechanisms of dual ANG-2 functions are poorly understood. Here, we identify a model for the opposing roles of ANG-2 in angiogenesis. We found that angiogenesis-activated endothelium harbored a subpopulation of TIE2-negative ECs (TIE2lo). TIE2 expression was downregulated in angiogenic ECs, which abundantly expressed several integrins. ANG-2 bound to these integrins in TIE2lo ECs, subsequently inducing, in a TIE2-independent manner, phosphorylation of the integrin adaptor protein FAK, resulting in RAC1 activation, migration, and sprouting angiogenesis. Correspondingly, in vivo ANG-2 blockade interfered with integrin signaling and inhibited FAK phosphorylation and sprouting angiogenesis of TIE2lo ECs. These data establish a contextual model whereby differential TIE2 and integrin expression, binding, and activation control the role of ANG-2 in angiogenesis. The results of this study have immediate translational implications for the therapeutic exploitation of angiopoietin signaling.

A novel player in cellular hypertrophy: Gißγ/PI3K-dependent activation of the RacGEF TIAM-1 is required for α1-adrenoceptor induced hypertrophy in neonatal rat cardiomyocytes.

Activation of α(1)-adrenoceptors (α(1)-AR) by high catecholamine levels, e.g. in heart failure, is thought to be a driving force of cardiac hypertrophy. In this context several downstream mediators and cascades have been identified to potentially play a role in cardiomyocyte hypertrophy. One of these proteins is the monomeric G protein Rac1. However, until now it is unclear how this essential G protein is activated by α(1)-AR agonists and what are the downstream targets inducing cellular growth. By using protein-based as well as pharmacological inhibitors and the shRNA technique, we demonstrate that in neonatal rat cardiomyocytes (NRCM) Rac1 is activated via a cascade involving the α(1A)-AR subtype, G(i)ßγ, the phosphoinositide-3'-kinase and the guanine nucleotide exchange factor Tiam1. We further demonstrate that this signaling induces an increase in protein synthesis, cell size and atrial natriuretic peptide expression. We identified the p21-activated kinase 2 (PAK2) as a downstream effector of Rac1 and were able to link this cascade to the activation of the pro-hypertrophic kinases ERK1/2 and p90RSK. Our data thus reveal a prominent role of the α(1A)-AR/G(i)ßγ/Tiam1-mediated activation of Rac1 and its effector PAK2 in the induction of hypertrophy in NRCM.