Background—: Delayed afterdepolarizations (DADs) carried by Na ⁺ -Ca ²⁺ -exchange current (I _NCX ) in response to sarcoplasmic reticulum (SR) Ca ²⁺ leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined. Methods and Results—: Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca ²⁺ ] _i (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca ²⁺ ] _i and SR Ca ²⁺ content (integrated I _NCX during caffeine-induced Ca ²⁺ transient) were unchanged, whereas diastolic SR Ca ²⁺ leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was ≈50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca ²⁺ leak. Calmodulin expression (60%), Ca ²⁺ /calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca ²⁺ leak, the frequency of spontaneous Ca ²⁺ release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca ²⁺ sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca ²⁺ ] _i and I _NCX density revealed I _NCX upregulation in cAF. Spontaneous Ca ²⁺ release events accompanied by inward I _NCX currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca ²⁺ ] _i (diastolic [Ca ²⁺ ] _i –voltage coupling gain) was higher in cAF compared with control. Conclusions—: Enhanced SR Ca ²⁺ leak through CaMKII-hyperphosphorylated RyR2, in combination with larger I _NCX for a given SR Ca ²⁺ release and increased diastolic [Ca ²⁺ ] _i -voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

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 (TIE2^lo). TIE2 expression was downregulated in angiogenic ECs, which abundantly expressed several integrins. ANG-2 bound to these integrins in TIE2^lo 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 TIE2^lo 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 cluster of manuscripts in this issue of the Journal highlights a lack of selectivity of 49 antibodies against 19 subtypes of α₁- and β-adrenoceptors, muscarinic, dopamine and galanin receptors as well as vanilloid (TRPV1) receptors. Taken together these data demonstrate that lack of selectivity appears to be the rule rather than the exception for antibodies against G-protein-coupled and perhaps also other receptors. Thus, the previously often applied validation of such antibodies by the disappearance of staining in the presence of blocking peptide, i.e. the antigen against which the antibody was raised, alone is insufficient to demonstrate specificity. We propose that receptor antibodies should be validated by at least one of the following techniques: a) disappearance of staining in knock-out animals of the target receptor, b) reduction of staining upon knock-down approaches such as siRNA treatment, c) selectivity of staining in immunoblots or immunocytochemistry for the target receptor vs. related subtypes when expressed in the same cell line and/or d) antibodies raised against multiple distinct epitopes of a receptor yielding very similar staining patterns. Other issues of consideration to obtain reliable results based on receptor antibodies in applications such as immunohistochemistry or immunoblotting are also being discussed.

Dilated cardiomyopathies (DCM) show remarkable variability in their age of onset, phenotypic presentation, and clinical course. Hence, disease mechanisms must exist that modify the occurrence and progression of DCM, either by genetic or epigenetic factors that may interact with environmental stimuli. In the present study, we examined genome‐wide cardiac DNA methylation in patients with idiopathic DCM and controls. We detected methylation differences in pathways related to heart disease, but also in genes with yet unknown function in DCM or heart failure, namely Lymphocyte antigen 75 ( LY75 ), Tyrosine kinase‐type cell surface receptor HER3 ( ERBB3 ), Homeobox B13 ( HOXB13 ) and Adenosine receptor A2A ( ADORA2A ). Mass‐spectrometric analysis and bisulphite‐sequencing enabled confirmation of the observed DNA methylation changes in independent cohorts. Aberrant DNA methylation in DCM patients was associated with significant changes in LY75 and ADORA2A mRNA expression, but not in ERBB3 and HOXB13 . In vivo studies of orthologous ly75 and adora2a in zebrafish demonstrate a functional role of these genes in adaptive or maladaptive pathways in heart failure.

The guanine nucleotide exchange factor p63RhoGEF is an effector of the heterotrimeric guanine nucleotide–binding protein (G protein) Gα _q and thereby links Gα _q -coupled receptors (GPCRs) to the activation of the small-molecular-weight G protein RhoA. We determined the crystal structure of the Gα _q -p63RhoGEF-RhoA complex, detailing the interactions of Gα _q with the Dbl and pleckstrin homology (DH and PH) domains of p63RhoGEF. These interactions involve the effector-binding site and the C-terminal region of Gα _q and appear to relieve autoinhibition of the catalytic DH domain by the PH domain. Trio, Duet, and p63RhoGEF are shown to constitute a family of Gα _q effectors that appear to activate RhoA both in vitro and in intact cells. We propose that this structure represents the crux of an ancient signal transduction pathway that is expected to be important in an array of physiological processes.

Rationale:: Increased activity of Ca ²⁺ /calmodulin-dependent protein kinase II (CaMKII) is thought to promote heart failure (HF) progression. However, the importance of CaMKII phosphorylation of ryanodine receptors (RyR2) in HF development and associated diastolic sarcoplasmic reticulum Ca ²⁺ leak is unclear. Objective:: Determine the role of CaMKII phosphorylation of RyR2 in patients and mice with nonischemic and ischemic forms of HF. Methods and Results:: Phosphorylation of the primary CaMKII site S2814 on RyR2 was increased in patients with nonischemic, but not with ischemic, HF. Knock-in mice with an inactivated S2814 phosphorylation site were relatively protected from HF development after transverse aortic constriction compared with wild-type littermates. After transverse aortic constriction, S2814A mice did not exhibit pulmonary congestion and had reduced levels of atrial natriuretic factor. Cardiomyocytes from S2814A mice exhibited significantly lower sarcoplasmic reticulum Ca ²⁺ leak and improved sarcoplasmic reticulum Ca ²⁺ loading compared with wild-type mice after transverse aortic constriction. Interestingly, these protective effects on cardiac contractility were not observed in S2814A mice after experimental myocardial infarction. Conclusions:: Our results suggest that increased CaMKII phosphorylation of RyR2 plays a role in the development of pathological sarcoplasmic reticulum Ca ²⁺ leak and HF development in nonischemic forms of HF such as transverse aortic constriction in mice.