Issam Abu-Taha

Enhanced Sarcoplasmic Reticulum Ca2+ Leak and Increased Na+-Ca2+ Exchanger Function Underlie Delayed Afterdepolarizations in Patients With Chronic Atrial Fibrillation

Background— Delayed afterdepolarizations (DADs) carried by Na+-Ca2+-exchange current (INCX) in response to sarcoplasmic reticulum (SR) Ca2+ 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 [Ca2+]i (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca2+]i and SR Ca2+ content (integrated INCX during caffeine-induced Ca2+ transient) were unchanged, whereas diastolic SR Ca2+ 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 Ca2+ leak. Calmodulin expression (60%), Ca2+/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 Ca2+ leak, the frequency of spontaneous Ca2+ 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 Ca2+ sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca2+]i and INCX density revealed INCX upregulation in cAF. Spontaneous Ca2+ release events accompanied by inward INCX currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca2+]i (diastolic [Ca2+]i–voltage coupling gain) was higher in cAF compared with control. Conclusions— Enhanced SR Ca2+ leak through CaMKII-hyperphosphorylated RyR2, in combination with larger INCX for a given SR Ca2+ release and increased diastolic [Ca2+]i-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

The interaction of nucleoside diphosphate kinase B with Gβγ dimers controls heterotrimeric G protein function

Heterotrimeric G proteins in physiological and pathological processes have been extensively studied so far. However, little is known about mechanisms regulating the cellular content and compartmentalization of G proteins. Here, we show that the association of nucleoside diphosphate kinase B (NDPK B) with the G protein βγ dimer (Gβγ) is required for G protein function in vivo. In zebrafish embryos, morpholino-mediated knockdown of zebrafish NDPK B, but not NDPK A, results in a severe decrease in cardiac contractility. The depletion of NDPK B is associated with a drastic reduction in Gβ1γ2 dimer expression. Moreover, the protein levels of the adenylyl cyclase (AC)-regulating Gαs and Gαi subunits as well as the caveolae scaffold proteins caveolin-1 and -3 are strongly reduced. In addition, the knockdown of the zebrafish Gβ1 orthologs, Gβ1 and Gβ1like, causes a cardiac phenotype very similar to that of NDPK B morphants. The loss of Gβ1/Gβ1like is associated with a down-regulation in caveolins, AC-regulating Gα-subunits, and most important, NDPK B. A comparison of embryonic fibroblasts from wild-type and NDPK A/B knockout mice demonstrate a similar reduction of G protein, caveolin-1 and basal cAMP content in mammalian cells that can be rescued by re-expression of human NDPK B. Thus, our results suggest a role for the interaction of NDPK B with Gβγ dimers and caveolins in regulating membranous G protein content and maintaining normal G protein function in vivo.

Through scaffolding and catalytic actions nucleoside diphosphate kinase B differentially regulates basal and β-adrenoceptor-stimulated cAMP synthesis

β-adrenoceptors (βAR) play a central role in the regulation of cAMP synthesis and cardiac contractility. Nucleoside diphosphate kinase B (NDPK B) regulates cAMP signalling by complex formation with Gβγ dimers thereby activating and stabilizing heterotrimeric G(s) proteins, key transducer of βAR signals into the cell. Here, we explored the requirement of NDPK B for basal and βAR-stimulated cAMP synthesis and analysed the underlying mechanisms by comparing wild-type NDPK B (WT) and its catalytically inactive H118N mutant. Stable overexpression of both WT- and H118N-NDPK B in cardiomyocyte derived H10 cells increased the plasma membrane content of G(s) and caveolin-1 and thus enhanced the isoproterenol (ISO)-stimulated cAMP-synthesis by about 2-fold. Conversely, the loss of NDPK B in embryonic fibroblasts from NDPK A/B-depleted mice was associated with a severe reduction in membranous G(s) protein and carveolin-1 content causing a marked decrease in basal and ISO-induced cAMP formation. Re-expression of NDPK B, but not of NDPK A, was able to rescue this phenotype. Both, re-expression of WT- and H118N-NDPK B induced the re-appearance of G(s) and caveolin-1 at the plasma membrane to a similar extent. Accordingly, WT- and H118N-NDPK B similarly enhanced ISO-induced cAMP formation. In contrast, the catalytically inactive H118N-NDPK B was less potent and less effective in rescuing basal cAMP production. Identical results were obtained in neonatal rat cardiac myocytes after siRNA-induced knockdown and adenoviral re-expression of NDPK B. Our data reveal that NDPK B regulates G(s) function by two different mechanisms. The complex formation of NDPK B with G(s) is required for the stabilization of the G protein content at the plasma membrane. In addition, the NDPK B-dependent phosphotransfer reaction, which requires the catalytic activity, specifically allows a receptor-independent, basal G(s) activation.

Constitutive Activity of the Acetylcholine-Activated Potassium Current IK,ACh in Cardiomyocytes

Stimulation of the vagal nerve slows the heart rate and leads to shorter action potential duration in the atria. These effects are mainly mediated by binding of the vagal neurotransmitter acetylcholine to muscarinic type 2 receptors resulting in dissociation of Gi proteins and subsequent activation of IK,ACh-K(+) channels due to binding of Gβγ-subunits. Even though agonist-independent (constitutive) IK,ACh activity is considered negligible in the healthy heart, constitutive IK,ACh activity has been shown to contribute to remodeling processes associated with cardiac diseases such as atrial fibrillation. In this review, we summarize possible mechanisms, which may contribute to the development of constitutively active IK,ACh. For example, an increased availability of Gβγ-subunits within the IK,ACh channel complex could contribute to receptor-independent IK,ACh activation. Accordingly, reduced expression of Gα-subunits, which act as Gβγ-scavengers within the channel complex, and increased activity of nucleoside diphosphate kinases, which activate G proteins in a receptor-independent manner, are likely contributors to constitutively active IK,ACh. In addition, alterations of the IK,ACh channel composition or phosphorylation may also be involved in abnormal IK,ACh current activity. Finally, we discuss possible therapeutic applications of pharmacological IK,ACh modulators, which may represent future drug targets against cardiac diseases such as atrial fibrillation.

Enhanced Cardiomyocyte NLRP3 Inflammasome Signaling Promotes Atrial Fibrillation

Background: Atrial fibrillation (AF) is frequently associated with enhanced inflammatory response. The NLRP3 (NACHT, LRR, and PYD domain containing protein 3) inflammasome mediates caspase-1 activation and interleukin-1&bgr; release in immune cells but is not known to play a role in cardiomyocytes (CMs). Here, we assessed the role of CM NLRP3 inflammasome in AF. Methods: NLRP3 inflammasome activation was assessed by immunoblot in atrial whole-tissue lysates and CMs from patients with paroxysmal AF or long-standing persistent (chronic) AF. To determine whether CM-specific activation of NLPR3 is sufficient to promote AF, a CM-specific knockin mouse model expressing constitutively active NLRP3 (CM-KI) was established. In vivo electrophysiology was used to assess atrial arrhythmia vulnerability. To evaluate the mechanism of AF, electric activation pattern, Ca2+ spark frequency, atrial effective refractory period, and morphology of atria were evaluated in CM-KI mice and wild-type littermates. Results: NLRP3 inflammasome activity was increased in the atrial CMs of patients with paroxysmal AF and chronic AF. CM-KI mice developed spontaneous premature atrial contractions and inducible AF, which was attenuated by a specific NLRP3 inflammasome inhibitor, MCC950. CM-KI mice exhibited ectopic activity, abnormal sarcoplasmic reticulum Ca2+ release, atrial effective refractory period shortening, and atrial hypertrophy. Adeno-associated virus subtype-9–mediated CM-specific knockdown of Nlrp3 suppressed AF development in CM-KI mice. Finally, genetic inhibition of Nlrp3 prevented AF development in CREM transgenic mice, a well-characterized mouse model of spontaneous AF. Conclusions: Our study establishes a novel pathophysiological role for CM NLRP3 inflammasome signaling, with a mechanistic link to the pathogenesis of AF, and establishes the inhibition of NLRP3 as a potential novel AF therapy approach.

Rhythm Control of Atrial Fibrillation in Heart Failure

Atrial fibrillation (AF) and heart failure (HF) are common cardiovascular pathologies with severe prognostic implications that show bidirectional interactions. Rate and rhythm control are the main therapeutic strategies for patients with AF and HF. There is a paucity of safe and effective antiarrhythmic drugs for rhythm control of AF in HF, with amiodarone and (in the United States) dofetilide as the only imperfect options. The basic mechanisms of AF are discussed and the evidence and limitations of AF rhythm control options for patients with HF are reviewed. In addition, novel potential antiarrhythmic strategies for rhythm control of AF are highlighted.

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

Background: Chronic heart failure (HF) is associated with altered signal transduction via &bgr;-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 G&agr;i2 was increased whereas the NDPK-C/G&agr;s 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 &bgr;-adrenoceptor/cAMP signaling and cardiac contractility. By switching from G&agr;s to G&agr;i2 activation, NDPK-C may contribute to lower cAMP levels and the related contractile dysfunction in HF.

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.

Targeting altered Nme heterooligomerization in disease

The enzymatic activity of nucleoside diphosphate kinases (NDPK), which are encoded by members of the nme gene family, has been discovered in the 1950s. It removes the terminal phosphate from a nucleoside triphosphate (NTP) and adds it to a nucleoside diphosphate (NDP) and thus it is important for nucleotide homeostasis in every living cell. At least the four group I Nme proteins (Nme1 Nme4) carry this enzymatic activity. Nme1 and Nme2 are most abundant and ubiquitously expressed. In the 1990s it became evident that enhanced cancer metastasis was linked to reduced expression of Nme1. Metastasis, the colonization of distant sites by a tumor, is often more life threatening than the growth of the primary tumor, which can be surgically removed in many cases. Thus, the identification of the function of Nme1 underlying its metastasis suppressor activity is a long standing goal in research [1]. It turned out that Nmes are “sticky” proteins. They obviously are not only found in many protein complexes but also bind to DNA and lipids. Thus a huge variety of functions have been attributed to Nme proteins, ranging from NTP supply, protein histidine kinase activity, exonuclease activity on DNA and protein scaffold as well as scavenger roles [2]. Therefore, the unique and to be targeted function of the Nme1 metastasis suppressor most likely does not exist. Recent work on Nme2 and Nme3 shed however light on an aspect nearly neglected so far in Nme research, heteroligomerization. Eukaryotic Nmes of the class I family form hexamers consistent of six enzymatically active monomers. Both, homoand heterohexamers have been described [3]. Previous work from our laboratory and others linked Nme2 to heterotrimeric G protein activation, again acting as NDPK for GTP supply, as protein histidine kinase on the G protein β-subunit and as a scaffold organizing G protein-mediated signal transduction in caveolae [4]. Recently, we established that the complex formation with the G proteins Gs and Gi Nme2/Nme3 requires heterooligomerization [5]. In cardiomyocytes, the Nme2/ Nme3 oligomers obviously regulate not only the activation of G proteins but also their abundance at the plasma membrane and their accessibility by G protein coupled receptors. Interestingly, the preference of the interaction of the Nme2/Nme3 complexes with the G proteins switches during the development of end-stage heart failure, a condition, at which the expression of Nme3 and Gi proteins is additionally upregulated. Whereas in healthy human heart Nme2/Nme3 oligomers are preferentially bound to Gs, in end-stage heart failure Gi2 was found predominantly in the complex. This switch has profound consequences on the resulting signal transduction (Figure 1). An increased complex formation of Nme2/Nme3 with Gs increases cAMP formation and contractility. In contrast, if Gi2 is bound, cAMP formation is constitutively suppressed which is in accordance with the observations from failing human hearts. Taken together, the data showed that the composition and localization of G protein signaling complexes can change over time during disease development and in response to altered Nme heteroligomerization. As Nme1 has also been shown to form heterooligomers at least with Nme2 [3], it is worthwhile to speculate that the composition of this heterohexamers has influence on the interaction and localization properties and is thus involved in the multifaceted actions also of this protein. As for Nme2, the exact composition of the Nme1 interactome under specific experimental and/or clinical conditions is still unknown. It can however be expected that the advances in super-resolution live-cell imaging methodologies will help to elucidate the subcellular localization of signaling complexes and their organization in near future. Insights into the temporal dynamics of the Nme oligomer composition and their interactome will certainly help to understand the function of this enzyme in Editorial

Profibrotic, Electrical, and Calcium-Handling Remodeling of the Atria in Heart Failure Patients With and Without Atrial Fibrillation

Atrial fibrillation (AF) and heart failure (HF) are common cardiovascular diseases that often co-exist. Animal models have suggested complex AF-promoting atrial structural, electrical, and Ca2+-handling remodeling in the setting of HF, but data in human samples are scarce, particularly regarding Ca2+-handling remodeling. Here, we evaluated atrial remodeling in patients with severe left ventricular (LV) dysfunction (HFrEF), long-standing persistent (‘chronic’) AF (cAF) or both (HFrEF-cAF), and sinus rhythm controls with normal LV function (Ctl) using western blot in right-atrial tissue, sharp-electrode action potential (AP) measurements in atrial trabeculae and voltage-clamp experiments in isolated right-atrial cardiomyocytes. Compared to Ctl, expression of profibrotic markers (collagen-1a, fibronectin, periostin) was higher in HFrEF and HFrEF-cAF patients, indicative of structural remodeling. Connexin-43 expression was reduced in HFrEF patients, but not HFrEF-cAF patients. AP characteristics were unchanged in HFrEF, but showed classical indices of electrical remodeling in cAF and HFrEF-cAF (prolonged AP duration at 20% and shorter AP duration at 50% and 90% repolarization). L-type Ca2+ current (ICa,L) was significantly reduced in HFrEF, cAF and HFrEF-cAF, without changes in voltage-dependence. Potentially proarrhythmic spontaneous transient-inward currents were significantly more frequent in HFrEF and HFrEF-cAF compared to Ctl, likely resulting from increased sarcoplasmic reticulum (SR) Ca2+ load (integrated caffeine-induced current) in HFrEF and increased ryanodine-receptor (RyR2) single-channel open probability in HFrEF and HFrEF-cAF. Although expression and phosphorylation of the SR Ca2+-ATPase type-2a (SERCA2a) regulator phospholamban were unchanged in HFrEF and HFrEF-cAF patients, protein levels of SERCA2a were increased in HFrEF-cAF and sarcolipin expression was decreased in both HFrEF and HFrEF-cAF, likely increasing SR Ca2+ uptake and load. RyR2 protein levels were decreased in HFrEF and HFrEF-cAF patients, but junctin levels were higher in HFrEF and relative Ser2814-RyR2 phosphorylation levels were increased in HFrEF-cAF, both potentially contributing to the greater RyR2 open probability. These novel insights into the molecular substrate for atrial arrhythmias in HF-patients position Ca2+-handling abnormalities as a likely trigger of AF in HF patients, which subsequently produces electrical remodeling that promotes the maintenance of the arrhythmia. Our new findings may have important implications for the development of novel treatment options for AF in the context of HF.