The inotropic and arrhythmogenic effects of acutely increased late INa are associated with elevated ROS but not oxidation of PKARIα.

Background: Acute stimulation of the late sodium current (INaL) as pharmacologically induced by Anemonia toxin II (ATX-II) results in Na+-dependent Ca2+ overload and enhanced formation of reactive oxygen species (ROS). This is accompanied by an acute increase in the amplitude of the systolic Ca2+ transient. Ca2+ transient amplitude is determined by L-type Ca2+-mediated transsarcolemmal Ca2+ influx (ICa) into the cytosol and by systolic Ca2+ release from the sarcoplasmic reticulum (SR). Type-1 protein kinase A (PKARIα) becomes activated upon increased ROS and is capable of stimulating ICa, thereby sustaining the amplitude of the systolic Ca2+ transient upon oxidative stress. Objectives: We aimed to investigate whether the increase of the systolic Ca2+ transient as acutely induced by INaL (by ATX-II) may involve stimulation of ICa through oxidized PKARIα. Methods: We used a transgenic mouse model in which PKARIα was made resistant to oxidative activation by homozygous knock-in replacement of redox-sensitive Cysteine 17 with Serine within the regulatory subunits of PKARIα (KI). ATX-II (at 1 nmol/L) was used to acutely enhance INaL in freshly isolated ventricular myocytes from KI and wild-type (WT) control mice. Epifluorescence and confocal imaging were used to assess intracellular Ca2+ handling and ROS formation. A ruptured-patch whole-cell voltage-clamp was used to measure INaL and ICa. The impact of acutely enhanced INaL on RIα dimer formation and PKA target structures was studied using Western blot analysis. Results: ATX-II increased INaL to a similar extent in KI and WT cells, which was associated with significant cytosolic and mitochondrial ROS formation in both genotypes. Acutely activated Ca2+ handling in terms of increased Ca2+ transient amplitudes and elevated SR Ca2+ load was equally present in KI and WT cells. Likewise, cellular arrhythmias as approximated by non-triggered Ca2+ elevations during Ca2+ transient decay and by diastolic SR Ca2+-spark frequency occurred in a comparable manner in both genotypes. Most importantly and in contrast to our initial hypothesis, ATX-II did not alter the magnitude or inactivation kinetics of ICa in neither WT nor KI cells and did not result in PKARIα dimerization (i.e., oxidation) despite a clear prooxidant intracellular environment. Conclusions: The inotropic and arrhythmogenic effects of acutely increased INaL are associated with elevated ROS, but do not involve oxidation of PKARIα.

Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in isolated mouse ventricular myocytes.

Background: The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib is associated with a higher incidence of cardiotoxic side effects including heart failure (HF). Objectives: Ibrutinib is capable of inhibiting PI3K/Akt signaling in neonatal rat ventricular cardiomyocytes when stimulated with insulin-like growth factor 1 (IGF-1). We therefore hypothesized that Ibrutinib might disrupt IGF-1-mediated activation of intracellular Ca handling in adult mouse cardiomyocytes by inhibiting PI3K/Akt signaling. Methods: Isolated ventricular myocytes (C57BL6/J) were exposed to IGF-1 at 10 nmol/L in the presence or absence of Ibrutinib (1 µmol/L) or Acalabrutinib (10 µmol/L; cell culture for 24 ± 2 h). Intracellular Ca handling was measured by epifluorescence (Fura-2 AM) and confocal microscopy (Fluo-4 AM). Ruptured-patch whole-cell voltage-clamp was used to measure ICa. Levels of key cardiac Ca handling proteins were investigated by immunoblots. Results: IGF-1 significantly increased Ca transient amplitudes by ∼83% as compared to vehicle treated control cells. This was associated with unaffected diastolic Ca, enhanced SR Ca loading and increased ICa. Co-treatment with Ibrutinib attenuated both the IGF-1-mediated increase in SR Ca content and in ICa. IGF-1 treated cardiomyocytes had significantly increased levels of pS473Akt/Akt and SERCA2a expression as compared to cells concomitantly treated with IGF-1 and Ibrutinib. SR Ca release (as assessed by Ca spark frequency) was unaffected by either treatment. In order to test for potential off-target effects, second generation BTK inhibitor Acalabrutinib with greater BTK selectivity and lower cardiovascular toxicity was tested for IGF1-mediated activation of intracellular Ca handling. Acalabrutinib induced similar effects on Ca handling in IGF-1 treated cultured myocytes as Ibrutinib in regard to decreased Ca transient amplitude and slowed Ca transient decay, hence implying a functional class effect of BTK inhibitors in cardiac myocytes. Conclusions: Inhibition of BTK by Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in adult ventricular mouse myocytes in the face of disrupted Akt signaling and absent SERCA2a upregulation.

A Pilot Trial to Compare the Long-Term Efficacy of Pulmonary Vein Isolation with High-Power Short-Duration Radiofrequency Versus Laser Energy with Rapid Ablation Mode.

BACKGROUND: Pulmonary vein (PV) reconnection is the major cause of atrial fibrillation (AF) recurrence after pulmonary vein isolation (PVI). The probability of reconnection is higher if the primary lesion is not sufficiently effective, which can be unmasked with an adenosine provocation test (APT). High-power short-duration radiofrequency energy (HPSD) guided with ablation index (AI) and the third generation of the visually guided laser balloon (VGLB) are new methods for PVI. METHODS: A total of 70 participants (35 in each group) who underwent a PVI with either AI-guided HPSD (50 W; AI 500 for the anterior and 400 for the posterior wall, respectively) or VGLB ablation were included in this observational pilot trial. Twenty minutes after each PVI, an APT was performed. The primary endpoint was the event-free survival from AF after three years. RESULTS: A total of 137 (100%) PVs in the HPSD arm and 131 PVs (98.5%) in the VGLB arm were initially successfully isolated (p = 0.24). The overall procedure duration was similar in both arms (155 ± 39 in HPSD vs. 175 ± 58 min in VGLB, p = 0.191). Fluoroscopy time, left atrial dwelling time and duration from the first to the last ablation were longer in the VGLB arm (23 ± 8 vs. 12 ± 3 min, p < 0.001; 157 (111-185) vs. 134 (104-154) min, p = 0.049; 92(59-108) vs. 72 (43-85) min, p = 0.010). A total of 127 (93%) in the HPSD arm and 126 (95%) PVs in the VGLB arm remained isolated after APT (p = 0.34). The primary endpoint was met 1107 ± 68 days after ablation in 71% vs. 66% in the VGLB and HPSD arms, respectively (p = 0.65). CONCLUSIONS: HPSD and VGLB did not differ with respect to long-term outcome of PVI. A large, randomized study should be conducted to compare clinical outcomes with respect to these new ablation techniques.

Enhanced Heart Failure in Redox-Dead Cys17Ser PKARIα Knock-In Mice.

Background PKARIα (protein kinase A type I-α regulatory subunit) is redox-active independent of its physiologic agonist cAMP. However, it is unknown whether this alternative mechanism of PKARIα activation may be of relevance to cardiac excitation-contraction coupling. Methods and Results We used a redox-dead transgenic mouse model with homozygous knock-in replacement of redox-sensitive cysteine 17 with serine within the regulatory subunits of PKARIα (KI). Reactive oxygen species were acutely evoked by exposure of isolated cardiac myocytes to AngII (angiotensin II, 1 µmol/L). The long-term relevance of oxidized PKARIα was investigated in KI mice and their wild-type (WT) littermates following transverse aortic constriction (TAC). AngII increased reactive oxygen species in both groups but with RIα dimer formation in WT only. AngII induced translocation of PKARI to the cell membrane and resulted in protein kinase A-dependent stimulation of ICa (L-type Ca current) in WT with no effect in KI myocytes. Consequently, Ca transients were reduced in KI myocytes as compared with WT cells following acute AngII exposure. Transverse aortic constriction-related reactive oxygen species formation resulted in RIα oxidation in WT but not in KI mice. Within 6 weeks after TAC, KI mice showed an enhanced deterioration of contractile function and impaired survival compared with WT. In accordance, compared with WT, ventricular myocytes from failing KI mice displayed significantly reduced Ca transient amplitudes and lack of ICa stimulation. Conversely, direct pharmacological stimulation of ICa using Bay K8644 rescued Ca transients in AngII-treated KI myocytes and contractile function in failing KI mice in vivo. Conclusions Oxidative activation of PKARIα with subsequent stimulation of ICa preserves cardiac function in the setting of acute and chronic oxidative stress.

Empagliflozin reduces Ca/calmodulin-dependent kinase II activity in isolated ventricular cardiomyocytes.

AIMS: The EMPA-REG OUTCOME study showed reduced mortality and hospitalization due to heart failure (HF) in diabetic patients treated with empagliflozin. Overexpression and Ca2+ -dependent activation of Ca2+ /calmodulin-dependent kinase II (CaMKII) are hallmarks of HF, leading to contractile dysfunction and arrhythmias. We tested whether empagliflozin reduces CaMKII- activity and improves Ca2+ -handling in human and murine ventricular myocytes. METHODS AND RESULTS: Myocytes from wild-type mice, mice with transverse aortic constriction (TAC) as a model of HF, and human failing ventricular myocytes were exposed to empagliflozin (1 µmol/L) or vehicle. CaMKII activity was assessed by CaMKII-histone deacetylase pulldown assay. Ca2+ spark frequency (CaSpF) as a measure of sarcoplasmic reticulum (SR) Ca2+ leak was investigated by confocal microscopy. [Na+ ]i was measured using Na+ /Ca2+ -exchanger (NCX) currents (whole-cell patch clamp). Compared with vehicle, 24 h empagliflozin exposure of murine myocytes reduced CaMKII activity (1.6 ± 0.7 vs. 4.2 ± 0.9, P < 0.05, n = 10 mice), and also CaMKII-dependent ryanodine receptor phosphorylation (0.8 ± 0.1 vs. 1.0 ± 0.1, P < 0.05, n = 11 mice), with similar results upon TAC. In murine myocytes, empagliflozin reduced CaSpF (TAC: 1.7 ± 0.3 vs. 2.5 ± 0.4 1/100 µm-1  s-1 , P < 0.05, n = 4 mice) but increased SR Ca2+ load and Ca2+ transient amplitude. Importantly, empagliflozin also significantly reduced CaSpF in human failing ventricular myocytes (1 ± 0.2 vs. 3.3 ± 0.9, P < 0.05, n = 4 patients), while Ca2+ transient amplitude was increased (F/F0 : 0.53 ± 0.05 vs. 0.36 ± 0.02, P < 0.05, n = 3 patients). In contrast, 30 min exposure with empagliflozin did not affect CaMKII activity nor Ca2+ -handling but significantly reduced [Na+ ]i . CONCLUSIONS: We show for the first time that empagliflozin reduces CaMKII activity and CaMKII-dependent SR Ca2+ leak. Reduced Ca2+ leak and improved Ca2+ transients may contribute to the beneficial effects of empagliflozin in HF.

SR Ca2+-leak and disordered excitation-contraction coupling as the basis for arrhythmogenic and negative inotropic effects of acute ethanol exposure.

AIMS: Ethanol has acute negative inotropic and arrhythmogenic effects. The underlying mechanisms, however, are largely unknown. Sarcoplasmic reticulum Ca2+-leak is an important mechanism for reduced contractility and arrhythmias. Ca2+-leak can be induced by oxidative stress and Ca2+/Calmodulin-dependent protein kinase II (CaMKII). Therefore, we investigated the influence of acute ethanol exposure on excitation-contraction coupling in atrial and ventricular cardiomyocytes. METHODS AND RESULTS: Isolated human atrial and murine atrial or ventricular cardiomyocytes were preincubated for 30 min and then superfused with control solution or solution containing ethanol. Ethanol had acute negative inotropic and positive lusitropic effects in human atrial muscle strips and murine ventricular cardiomyocytes. Accordingly, Ca2+-imaging indicated lower Ca2+-transient amplitudes and increased SERCA2a activity, while myofilament Ca2+-sensitivity was reduced. SR Ca2+-leak was assessed by measuring Ca2+-sparks. Ethanol induced severe SR Ca2+-leak in human atrial cardiomyocytes (calculated leak: 4.60 ±â€¯0.45 mF/F0 vs 1.86 ±â€¯0.26 in control, n ≥ 80). This effect was dose-dependent, while spontaneous arrhythmogenic Ca2+-waves increased ~5-fold, as investigated in murine cardiomyocytes. Delayed afterdepolarizations, which can result from increased SR Ca2+-leak, were significantly increased by ethanol. Measurements using the reactive oxygen species (ROS) sensor CM-H2DCFDA showed increased ROS-stress in ethanol treated cells. ROS-scavenging with N-acetylcysteine prevented negative inotropic and positive lusitropic effects in human muscle strips. Ethanol-induced Ca2+-leak was abolished in mice with knockout of NOX2 (the main source for ROS in cardiomyocytes). Importantly, mice with oxidation-resistant CaMKII (Met281/282Val mutation) were protected from ethanol-induced Ca2+-leak. CONCLUSION: We show for the first time that ethanol acutely induces strong SR Ca2+-leak, also altering excitation-contraction coupling. Acute negative inotropic effects of ethanol can be explained by reduced systolic Ca2+-release. Mechanistically, ROS-production via NOX2 and oxidative activation of CaMKII appear to play central roles. This provides a mechanism for the arrhythmogenic and negative inotropic effects of ethanol and suggests a druggable target (CaMKII).

Comparison of diverse platelet activation markers as indicators for left atrial thrombus in atrial fibrillation.

Atrial fibrillation (AF) is well known for being a major risk factor of thromboembolic stroke. We could recently demonstrate an association of monocyte-platelet aggregates (MPAs) with the degree of thrombogenicity in patients with AF. This study investigated platelet activation markers, as potential biomarkers for the presence of left atrial (LA) thrombus in patients with AF. One hundred and eight patients with symptomatic AF underwent transesophageal echocardiography (TEE) before scheduled cardioversion or pulmonary vein isolation. In order to determine the content of MPAs by flow-cytometric quantification analyses, blood was drawn on the day of TEE. The soluble CD40 Ligand (sCD40L) and soluble P-selectin (sP-selectin) were obtained by Cytometric Bead Arrays (CBA). D-dimer levels were detected by quantitative immunological determination of fibrin degradation products. Clinical, laboratory, and echocardiographic standard parameters were obtained from all patients, including the determination of the flow in the left atrial appendage (LAA). Patients with detected LA thrombus (n = 28) compared with patients without thrombus (n = 80) showed an increased number of common risk factors, such as age, diabetes, heart failure, and coronary artery disease (CAD). The presence of LA thrombus was associated with significantly increased levels of MPAs (147 ± 12 vs. 304 ± 29 per µl; p < 0.00), sCD40L (106.3 ± 31.0 vs. 33.5 ± 2.1 pg/ml, p = 0.027), and D-dimer (0.13 ± 0.02 vs. 0.69 ± 0.21 mg FEU/l, p = 0.015). In contrast, sP-selectin showed no association with LA thrombus. A multivariate regression analysis showed that MPAs, sCD40L as well as D-dimers were independent indicators for the existence of LA thrombus. MPAs above 170 cells/µl indicated LA thrombus with a high sensitivity of 93% and a specificity of 73% (OR 62, 95% CI. 6.9-557.2, p < 0.001) in patients with AF, whereas the D-dimer lost their quality as independent indicator by using the conventional cut-off of 0.5 mg/l within the regression analysis. MPAs, as well as the D-dimer, correlated significantly negatively with the flow in the LAA measured during TEE. The content of MPAs, sCD40L, and D-dimer, but not sP-selectin showed an increased dependence on LA thrombus in patients with AF. In our study group, MPAs showed the best diagnostic test accuracy of the compared platelet markers. The different results of the examined platelet activation markers could be an indication of diverse mechanisms of LA thrombus in AF. Further studies should evaluate whether determination of MPAs in clinical routine may suffice to indicate the presence of LA thrombus in patients with AF.

Reduction of atrial fibrillation burden by pulmonary vein isolation leads to a decrease of CD11b expression on inflammatory cells.

Aims: It is hypothesized that inflammation could promote structural and electrical remodelling processes in atrial fibrillation (AF). Atrial infiltration of monocytes and granulocytes has been shown to be dependent on CD11b expression. The aim of this study was to investigate whether treatment of AF by pulmonary vein isolation (PVI) may lead to reduced inflammation, as indicated by a decrease of CD11b expression on monocytes and granulocytes. Methods and results: Flow-cytometric quantification analysis and determination of systemic inflammatory markers of peripheral blood were performed in 75 patients undergoing PVI 1 day before and 6 months after PVI. The extent of activation of monocytes and granulocytes was measured by quantifying the cell adhesion molecule CD11b. The mean expression of CD11b on monocytes (20.9 ± 2.5 vs. 10.2 ± 1.4; P < 0.001) and granulocytes (13.9 ± 1.6 vs. 6.8 ± 0.5; P < 0.001), as well as the relative count of CD11b-positive monocytes (P < 0.05) and CD11b-positive granulocytes (P < 0.01) were significantly reduced when comparing the identical patients before and 6 months after PVI. Systemic inflammatory parameters showed only a declining tendency after 6 months. Patients with unsuccessful PVI and ongoing AF on the day of follow-up showed no decrease in CD11b expression. Conclusions: A significant reduction of CD11b expression on monocytes and granulocytes, as a sign of reduced cellular inflammation, was achieved by treatment of AF using PVI. These data strongly support that AF is not only a consequence of but also a cause for inflammatory processes, which, in turn, may contribute to atrial remodelling.

Association of platelet activation markers with recurrence of atrial fibrillation after pulmonary vein isolation.

Atrial fibrillation (AF) is known to cause platelet activation. AF and its degree of thrombogenesis could be associated with monocyte-platelet aggregates (MPAs). We investigated on whether the content of MPAs or other platelet activation markers is associated with the recurrence of AF after pulmonary vein isolation (PVI). A total of 73 patients with symptomatic AF underwent PVI. After 6 months, all patients were evaluated for episodes of AF recurrence. At the same time, flow-cytometric quantification analyses were performed to determine the content of MPAs. Further platelet activation parameters were detected by using either cytometric bead arrays or quantitative immunological determination. Patients with recurrent AF (n = 20) compared to individuals without AF relapse (n = 53) were associated with an increased content of MPAs (43 ± 3% vs. 33 ± 2%, p = 0.004), as well as an increased CD41 expression on monocytes (191 ± 20 vs. 113 ± 6, p = 0.001). The level of the soluble platelet activation markers such as D-dimer, sCD40L, and sP-selectin did not differ between these groups. The content of MPAs correlated weakly with the level of sCD40L (r = 0.26, p = 0.03), but not with sP-selectin and D-dimer, whereas sP-selectin and sCD40L correlated with each other (r = 0.38, p = 0.001). Only the cellular marker of platelet activation, the content of MPAs, was increased in patients with recurrent AF after PVI. In contrast, soluble markers remained unaltered. These data indicate a distinct mechanism and level of platelet activation in AF. The clinical relevance of MPAs in identifying AF recurrence or in guiding the therapy with anticoagulants remains to be elucidated.

Atrial fibrillation is associated with high levels of monocyte-platelet-aggregates and increased CD11b expression in patients with aortic stenosis.

A growing body of evidence suggests a pivotal role of inflammatory processes in AF in a bidirectional manner. Infiltrating leukocytes seem to promote both structural and electrical remodelling processes in patients with AF. Monocyte-platelets-aggregates (MPAs) are sensitive markers of both platelets and monocyte activation. So far it is not clear whether the content of MPAs is affected by AF. The present study examined the content of MPAs and the activation of monocytes in elderly patients with an aortic stenosis in dependence of AF. These patients are known to have a high prevalence of AF. Flow-cytometric quantification analysis demonstrated that patients with AF have an increased content of MPAs (207 ± 13 cells/µl vs 307 ± 21 cells/µl, p< 0.001), and enhanced expression of CD11b on monocytes (p< 0.001), compared to patients in stable sinus rhythm (SR). The number of CD14+/CD16+ monocytes were only slightly elevated in patients with AF. These findings were seen in patients with permanent AF. But also patients with paroxysmal AF, even when presenting in SR, the MPAs were increased by 50 % (p< 0.05) as well as the CD11b expression, which was twice as high (p< 0.05) compared to stable SR. These results demonstrate for the first time a dependency of MPAs and CD11b expression on monocytes in the presence of AF and support the notion of a close relationship between AF, thrombogenesis and inflammation. The content of MPAs and the extent of activation on monocytes appear promising as biomarkers for paroxysmal AF and as possible future targets for developing novel pharmacological therapeutic strategies.

Monocyte-platelet aggregates and CD11b expression as markers for thrombogenicity in atrial fibrillation.

BACKGROUND: A strong interdependence is known between atrial fibrillation (AF), inflammation and thrombogenesis. Monocyte-platelet aggregates (MPAs) are sensitive markers of platelets and monocyte activation. It is not known whether MPAs are associated with thrombogenicity in AF. Therefore, we examined differences in the content of MPAs and CD11b expression in patients with AF in dependence of the presence of atrial thrombus formation. METHODS: 107 patients with symptomatic AF underwent transesophageal echocardiography (TEE) before planned cardioversion or pulmonary vein isolation. Flow-cytometric quantification analysis was done on the day of performed TEE to determine the content of MPAs and the expression of CD11b on monocytes and granulocytes. RESULTS: Compared to patients without thrombus (n = 80) those with an echocardiographic proven left atrium (LA) thrombus (n = 27) showed an increased extent of the risk factors age, diabetes and heart failure. The content of MPAs (147 ± 12 vs. 311 ± 29 cells/µl, p < 0.001) as well as the CD11b expression on monocytes (p < 0.05) and granulocytes (p < 0.05) were strongly associated with the existence of a LA thrombus. The content of MPAs and the CD11b expression remained independent predictors for LA thrombus after adjustment in logistic regression analysis and negatively correlated with left atrial appendage flow velocity. MPAs above 170 cells/µl (OR 34.2, p = 0.01) had a sensitivity of 96 % and a specificity of 73 % for predicting LA-thrombus. CONCLUSIONS: The content of MPAs and the CD11b expression on monocytes and granulocytes are increased in AF-patients with proven thrombus formation. They seem to be appropriate biomarkers for stratification of thromboembolic risk in patients with AF.