Analysis of the Effectiveness of the Latest 4th Generation Cryoballoon Catheters in Pulmonary Vein Isolation Using High-Resolution Mapping.

BACKGROUND: Long-term data showed that up to 27% of pulmonary veins are reconnected using cryoballoon ablation. This study aims to evaluate the efficacy of the latest 4th generation cryoballoon catheters using ultra high-resolution mapping. METHODS: In patients with AF, a standard PVI with the latest 4th generation cryoballoon catheter (Arctic Front Advance PRO, Medtronic Minneapolis, USA) and the spiral mapping catheter (Achieve Advance, Medtronic, Minneapolis, MN, USA) was performed. Subsequently, high-resolution mapping was achieved using the novel multipolar grid mapping catheter (Advisor HD Grid SE, Abbott Laboratories; USA). Follow-up was obtained after 6 months by means of a 7-day Holter ECG. RESULTS: In our study, acute PVI was successfully achieved in all 31 patients. The latest 4th generation cryoballoon catheter is safe in the acute phase of PVI. Additional high-resolution mapping (mean points per map 21001 ± 4911) using the multipolar grid mapping catheter enabled us to identify residual gaps only in the carina PV region; therefore, no additional ablation was performed. Three out of 31 patients (10%) presented with atrial arrhythmia recurrence always related with PV reconnection; using high-resolution mapping had no additional benefit in identifying pulmonary veins in which reconnection will occur. CONCLUSION: The utility of additional high-density mapping, facilitated by the HD Grid catheter following PVI with the fourth-generation cryoballoon catheter do not substantiate a discernible advantage over conventional mapping methodologies, particularly the spiral mapping catheter. Residual carinal conduction was observed in a substantial cohort of patients (48%), highlighting a persistent challenge in achieving complete electrical isolation.

The orphan receptor GPRC5B activates pro-inflammatory signaling in the vascular wall via Fyn and NFκB.

BACKGROUND AND AIMS: Atherosclerosis is driven by an inflammatory process of the vascular wall. The novel orphan G-protein coupled receptor 5B of family C (GPRC5B) is involved in drosophila sugar and lipid metabolism as well as mice adipose tissue inflammation. Here, we investigated the role of GPRC5B in the pro-atherogenic mechanisms of hyperglycemia and vascular inflammation. METHODS: Immortalized and primary endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) were used for stimulation with high glucose or different cytokines. Adenoviral- or plasmid-driven GPRC5B overexpression and siRNA-mediated knockdown were performed in these cells to analyze functional and mechanistic pathways of GPRC5B. RESULTS: In ECs and VSMCs, stimulation with high glucose, TNFα or LPS induced a significant upregulation of endogenous GPRC5B mRNA and protein levels. GPRC5B overexpression and knockdown increased and attenuated, respectively, the expression of the pro-inflammatory cytokines TNFα, IL-1ß, IL-6 as well as the pro-atherogenic vascular adhesion molecules ICAM-1 and VCAM-1. Furthermore, the expression and activity of the metalloproteinase MMP-9, a component of atherosclerotic plaque stabilization, were significantly enhanced by GPRC5B overexpression. Mechanistically, GPRC5B increased the phosphorylation of ERK1/2 and activated NFκB through a direct interaction with the tyrosine kinase Fyn. CONCLUSIONS: Our findings demonstrate that GPRC5B is upregulated in response to high glucose and pro-inflammatory signaling. GPRC5B functionally modulates the inflammatory activity in cells of the vascular wall, suggesting a pro-atherogenic GPRC5B-dependent positive feedback loop via Fyn and NFκB. Thus, GPRC5B warrants further attention as a novel pharmacological target for the treatment of vascular inflammation and possibly atherogenesis.

Renovascular hypertension: endovascular therapy in complicated aortic Stanford type B dissection.

A 63 years old male with a history of arterial hypertension presented with a current onset of chest pain and discrete headaches accompanied with dizziness. His blood pressure, 210/110 mmHg, had worsened and showed a reversed circadian rhythm with an average of 150/90 mmHg during night time. A CT angiography of the aorta demonstrated a type B dissection involving the right renal artery causing reduced perfusion of the right kidney. Subsequent invasive aortic angiography showed a continuously moving aortic dissection flap resulting in a dynamic stenosis proven by varying pressure gradients of between 5 and 35 mmHg. Stent placement of the renal artery ostium kept the vessel open and fixed the reno-aortic dissection flap in order to prevent it from progressing into the right kidney. Follow-up examinations revealed improved blood pressure control allowing for physiologic drop of blood pressure during night-time.

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.

Competition for Gßγ dimers mediates a specific cross-talk between stimulatory and inhibitory G protein α subunits of the adenylyl cyclase in cardiomyocytes.

Heterotrimeric G proteins are key regulators of signaling pathways in mammalian cells. Beyond G protein-coupled receptors, the amount and mutual ratio of specific G protein α, ß, and γ subunits determine the G protein signaling. However, little is known about mechanisms that regulate the concentration and composition of G protein subunits at the plasma membrane. Here, we show a novel cross-talk between stimulatory and inhibitory G protein α subunits (Gα) that is mediated by G protein ßγ dimers and controls the abundance of specific Gα subunits at the plasma membrane. Firstly, we observed in heart tissue from constitutively Gαi2- and Gαi3-deficient mice that the loss of Gαi2 and Gαi3 was accompanied by a slight increase in the protein content of the nontargeted Gαi isoform. Therefore, we analyzed whether overexpression of selected Gα subunits conversely impairs endogenous G protein α and ß subunit levels in cardiomyocytes. Integration of overexpressed Gαi2 subunits into heterotrimeric G proteins was verified by co-immunoprecipitation. Adenoviral expression of increasing amounts of Gαi2 led to a reduction of Gαi3 (up to 90 %) and Gαs (up to 75 %) protein levels. Likewise, increasing amounts of adenovirally expressed Gαs resulted in a linear 75 % decrease in both Gαi2 and Gαi3 protein levels. In contrast, overexpression of either Gαi or Gαs isoform did not influence the amount of Gαo and Gαq, both of which are not involved in the regulation of adenylyl cyclase activity. The mRNA expression of the disappearing endogenous Gα subunits was not affected, indicating a posttranslational mechanism. Interestingly, the amount of endogenous G protein ßγ dimers was not altered by any Gα overexpression. However, the increase of Gßγ level by adenoviral expression prevented the loss of endogenous Gαs and Gαi3 in Gαi2 overexpressing cardiomyocytes. Thus, our results provide evidence for a novel mechanism cross-regulating adenylyl cyclase-modulating Gαi isoforms and Gαs proteins. The Gα subunits apparently compete for a limited amount of Gßγ dimers, which are required for G protein heterotrimer formation at the plasma membrane.

Nucleoside diphosphate kinase B is required for the formation of heterotrimeric G protein containing caveolae.

Caveolae are flask-shaped invaginations in the plasma membrane that serve to compartmentalize and organize signal transduction processes, including signals mediated by G protein-coupled receptors and heterotrimeric G proteins. Herein we report evidence for a close association of the nucleoside diphosphate kinase B (NDPK B) and caveolin proteins which is required for G protein scaffolding and caveolae formation. A concomitant loss of the proteins NDPK B, caveolin isoforms 1 (Cav1) and 3, and heterotrimeric G proteins occurred when one of these proteins was specifically depleted in zebrafish embryos. Co-immunoprecipitation of Cav1 with the G protein Gß-subunit and NDPK B from zebrafish lysates corroborated the direct association of these proteins. Similarly, in embryonic fibroblasts from the respective knockout (KO) mice, the membrane content of the Cav1, Gß, and NDPK B was found to be mutually dependent on one another. A redistribution of Cav1 and Gß from the caveolae containing fractions of lower density to other membrane compartments with higher density could be detected by means of density gradient fractionation of membranes derived from NDPK A/B KO mouse embryonic fibroblasts (MEFs) and after shRNA-mediated NDPK B knockdown in H10 cardiomyocytes. This redistribution could be visualized by confocal microscopy analysis showing a decrease in the plasma membrane bound Cav1 in NDPK A/B KO cells and vice versa and a decrease in the plasma membrane pool of NDPK B in Cav1 KO cells. Consequently, ultrastructural analysis revealed a reduction of surface caveolae in the NDPK A/B KO cells. To prove that the disturbed subcellular localization of Cav1 in NDPK A/B KO MEFs as well as NDPK B in Cav1 KO MEFs is a result of the loss of NDPK B and Cav1, respectively, we performed rescue experiments. The adenoviral re-expression of NDPK B in NDPK A/B KO MEFs rescued the protein content and the plasma membrane localization of Cav1. The expression of an EGFP-Cav1 fusion protein in Cav1-KO cells induced a restoration of NDPK B expression levels and its appearance at the plasma membrane. We conclude from these findings that NDPK B, heterotrimeric G proteins, and caveolins are mutually dependent on each other for stabile localization and caveolae formation at the plasma membrane. The data point to a disturbed transport of caveolin/G protein/NDPK B complexes from intracellular membrane compartments if one of the components is missing.

The inotropic peptide ßARKct improves ßAR responsiveness in normal and failing cardiomyocytes through G(ßγ)-mediated L-type calcium current disinhibition.

RATIONALE: The G(ßγ)-sequestering peptide ß-adrenergic receptor kinase (ßARK)ct derived from the G-protein-coupled receptor kinase (GRK)2 carboxyl terminus has emerged as a promising target for gene-based heart failure therapy. Enhanced downstream cAMP signaling has been proposed as the underlying mechanism for increased ß-adrenergic receptor (ßAR) responsiveness. However, molecular targets mediating improved cardiac contractile performance by ßARKct and its impact on G(ßγ)-mediated signaling have yet to be fully elucidated. OBJECTIVE: We sought to identify G(ßγ)-regulated targets and signaling mechanisms conveying ßARKct-mediated enhanced ßAR responsiveness in normal (NC) and failing (FC) adult rat ventricular cardiomyocytes. METHODS AND RESULTS: Assessing viral-based ßARKct gene delivery with electrophysiological techniques, analysis of contractile performance, subcellular Ca²(+) handling, and site-specific protein phosphorylation, we demonstrate that ßARKct enhances the cardiac L-type Ca²(+) channel (LCC) current (I(Ca)) both in NCs and FCs on ßAR stimulation. Mechanistically, ßARKct augments I(Ca) by preventing enhanced inhibitory interaction between the α1-LCC subunit (Cav1.2α) and liberated G(ßγ) subunits downstream of activated ßARs. Despite improved ßAR contractile responsiveness, ßARKct neither increased nor restored cAMP-dependent protein kinase (PKA) and calmodulin-dependent kinase II signaling including unchanged protein kinase (PK)Cε, extracellular signal-regulated kinase (ERK)1/2, Akt, ERK5, and p38 activation both in NCs and FCs. Accordingly, although ßARKct significantly increases I(Ca) and Ca²(+) transients, being susceptible to suppression by recombinant G(ßγ) protein and use-dependent LCC blocker, ßARKct-expressing cardiomyocytes exhibit equal basal and ßAR-stimulated sarcoplasmic reticulum Ca²(+) load, spontaneous diastolic Ca²(+) leakage, and survival rates and were less susceptible to field-stimulated Ca²(+) waves compared with controls. CONCLUSION: Our study identifies a G(ßγ)-dependent signaling pathway attenuating cardiomyocyte I(Ca) on ßAR as molecular target for the G(ßγ)-sequestering peptide ßARKct. Targeted interruption of this inhibitory signaling pathway by ßARKct confers improved ßAR contractile responsiveness through increased I(Ca) without enhancing regular or restoring abnormal cAMP-signaling. ßARKct-mediated improvement of I(Ca) rendered cardiomyocytes neither susceptible to ßAR-induced damage nor arrhythmogenic sarcoplasmic reticulum Ca²(+) leakage.

Reversible histidine phosphorylation in mammalian cells: a teeter-totter formed by nucleoside diphosphate kinase and protein histidine phosphatase 1.

Regulation of protein phosphorylation by kinases and phosphatases is involved in many signaling pathways in mammalian cells. In contrast to prokaryotes and lower eukaryotes a role for the reversible phosphorylation of histidine residues is just emerging. The ß subunit of heterotrimeric G proteins, the metabolic enzyme adenosine 5'-triphosphate-citrate lyase (ACL), and the Ca2+-activated K+ channel KCa3.1 have been identified as targets for nucleoside diphosphate kinase (NDPK) acting as protein histidine kinase and the so far only identified mammalian protein histidine phosphatase (PHPT-1). Herein, we describe the analysis of the phosphorylation and dephosphorylation of histidine residues by NDPK and PHPT-1. In addition, experimental protocols for studying the consequences of heterotrimeric G protein activation via NDPK/Gßγ mediated phosphorelay, the regulation of ACL activity and of KCa3.1 conductivity by histidine phosphorylation will be presented.

Nucleoside diphosphate kinase-mediated activation of heterotrimeric G proteins.

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to receptor independent G protein activation. Apparently, the NDPK B isoform forms complexes with Gbetagamma dimers and thereby phosphorylates His266 in Gbeta1 subunits. Phosphorylated His266 mediates G protein activation by a transfer of the high energetic phosphate onto GDP, thus leading to de novo synthesis of GTP. Moreover, it has been demonstrated that the sarcolemmal content of NDPK isoforms is increased in hearts with terminal congestive heart failure leading to enhanced G protein activation. Similar data were reported in a rat model for beta-adrenoceptor-induced cardiac hypertrophy. We therefore describe in this chapter several methods which can be used for analysis of NDPK mediated G protein activation: (1) The quantification of NDPK isoforms in highly purified cardiac sarcolemmal membranes, (2) the enrichment of the NDPK B/Gbetagamma-complex from preparations of the retinal G protein transducin, (3) the analysis of the enhanced NDPK activated and high energy phosphate transfer in a neonatal rat cardiac myocyte derived cell line stably overexpressing NDPK (H10 cells), and (4) the increased activation of adenylyl cyclase by the enhanced receptor-independent activation of the stimulatory G protein alpha subunit in these cells.