BACKGROUND:Mitochondrial calcium (Ca2+) is a key regulator of cardiac energetics by stimulating the tricarboxylic acid cycle during elevated workload. Atrial fibrillation (AF) is associated with a reduction in cytosolic Ca2+ transient amplitude, but its effect on mitochondrial Ca2+ handling and cellular redox state has not been explored. METHODS:Cardiac myocytes isolated from patient-derived right atrial biopsies were subjected to workload transitions using patch-clamp stimulation and β-adrenergic stimulation (isoproterenol). In conjunction, nicotinamide adenine dinucleotide (phosphate)/flavin adenine dinucleotide (NAD[P]H/FAD) autofluorescence, cytosolic and mitochondrial [Ca2+] were monitored using epifluorescence microscopy. Sarcoplasmic reticulum and mitochondria were imaged using electron microscopy and tomography and stimulated emission depletion microscopy. The effects of the mitochondrial Ca2+ uptake enhancer ezetimibe on proarrhythmic activity in atrial myocytes and on AF burden in patients were investigated. RESULTS:Mitochondrial Ca2+ accumulation during increased workload was blunted in AF, and was associated with impaired regeneration of nicotinamide adenine dinucleotide and flavin adenine dinucleotide. Nanoscale imaging revealed spatial disorganization of sarcoplasmic reticulum and mitochondria, associated with microtubule destabilization. This was confirmed in human induced pluripotent stem cell-derived cardiac myocytes, where treatment with the microtubule destabilizer nocodazole displaced mitochondria and increased proarrhythmic Ca2+ sparks, which were rescued by MitoTEMPO. Ezetimibe also reduced the occurrence of arrhythmogenic Ca2+ release events both in AF myocytes and nocodazole-treated human induced pluripotent stem cell-derived cardiac myocytes. Retrospective patient analysis also revealed a reduced AF burden in patients on ezetimibe treatment. CONCLUSIONS:Mitochondrial Ca2+ uptake and accumulation are impaired in atrial myocytes from patients with AF. The disturbed spatial association between sarcoplasmic reticulum and mitochondria driven by destabilized microtubules may underlie impaired Ca2+ transfer in AF. Enhancing mitochondrial Ca2+ uptake potentially protects against arrhythmogenic events.

Blebbistatin is an excitation-contraction uncoupling agent commonly used in cardiac optical mapping; however, it has been reported to influence cardiac myofilament Ca2+ sensitivity. As primary contributors to Ca2+ buffering within cardiomyocytes, cardiac myofilaments play a critical role, and even minor disruptions in intracellular Ca2+ buffering significantly alter the free Ca2+ concentration. In this study, we investigated the effect of blebbistatin, a myosin II ATPase inhibitor, on intracellular Ca2+ buffering and cellular electrophysiology in induced pluripotent stem cell-derived atrial cardiomyocytes. Simultaneous whole-cell ruptured patch-clamp and fluorescence microscopy techniques were used to assess intracellular Ca2+ handling, in addition to automated high-throughput patch-clamp to investigate ion channel function. Comprehensive analysis of Ca2+ buffering revealed that blebbistatin (10 µmol/l) causes a significant increase in buffer dissociation constant, suggesting decreased affinity of Ca2+ buffers. Furthermore, systolic and diastolic Ca2+ levels, sarcoplasmic reticulum Ca2+ leak and the incidence of spontaneous Ca2+ release events were significantly higher upon blebbistatin treatment. Although there was lack of impact on INa and ICa,L peak density, Ca2+-dependent inactivation of ICa,L was significantly enhanced, and IK1 density was significantly smaller after blebbistatin. Importantly, these effects were reversed after chelation of intracellular Ca2+ with EGTA. Our observations indicate that blebbistatin reduces Ca2+ buffering, which, in turn, causes changes in cellular electrophysiology in cardiomyocytes. KEY POINTS: Intracellular Ca2+ buffering plays an important role in determining Ca2+ dynamics in cardiomyocytes. Blebbistatin, an excitation-contraction uncoupling agent widely used in experimental studies, decreases the affinity of intracellular Ca2+ buffers, as indicated by an increased buffer dissociation constant. Blebbistatin leads to higher systolic and diastolic Ca2+ levels and increased sarcoplasmic reticulum Ca2+ leak. Blebbistatin enhances L-type Ca2+ current (ICa,L) Ca2+-dependent inactivation and reduces inward rectifier potassium current (IK1) density, while INa and ICa,L remain unchanged. The effects of blebbistatin are mitigated by chelation of intracellular Ca2+ with EGTA, showing that they are secondary to altered Ca2+ buffering.

BACKGROUND:Alterations in the buffering of intracellular Ca2+, for which myofilament proteins play a key role, have been shown to promote cardiac arrhythmia. It is interesting that although studies report atrial myofibrillar degradation in patients with persistent atrial fibrillation (persAF), the intracellular Ca2+ buffering profile in persAF remains obscure. Therefore, we aimed to investigate the intracellular buffering of Ca2+ and its potential arrhythmogenic role in persAF.METHODS:Transmembrane Ca2+ fluxes (patch-clamp) and intracellular Ca2+ signaling (fluo-3-acetoxymethyl ester) were recorded simultaneously in myocytes from right atrial biopsies of sinus rhythm (Ctrl) and patients with persAF, alongside human atrial subtype induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs). Protein levels were quantified by immunoblotting of human atrial tissue and induced pluripotent stem cell-derived cardiac myocytes. Mouse whole heart and atrial electrophysiology were measured on a Langendorff system.RESULTS:Cytosolic Ca2+ buffering was decreased in atrial myocytes of patients with persAF because of a depleted amount of Ca2+ buffers. In agreement, protein levels of selected Ca2+ binding myofilament proteins, including cTnC (cardiac troponin C), a major cytosolic Ca2+ buffer, were significantly lower in patients with persAF. Small interfering RNA (siRNA)-mediated knockdown of cTnC (si-cTNC) in atrial iPSC-CM phenocopied the reduced cytosolic Ca2+ buffering observed in persAF. Si-cTnC treated atrial iPSC-CM exhibited a higher predisposition to spontaneous Ca2+ release events and developed action potential alternans at low stimulation frequencies. Last, indirect reduction of cytosolic Ca2+ buffering using blebbistatin in an ex vivo mouse whole heart model increased vulnerability to tachypacing-induced atrial arrhythmia, validating the direct mechanistic link between impaired cytosolic Ca2+ buffering and atrial arrhythmogenesis.CONCLUSIONS:Our findings suggest that loss of myofilament proteins, particularly reduced cTnC protein levels, causes diminished cytosolic Ca2+ buffering in persAF, thereby potentiating the occurrence of spontaneous Ca2+ release events and atrial fibrillation susceptibility. Strategies targeting intracellular buffering may represent a promising therapeutic lead in persAF management.

Background: Alternans, the periodic beat-to-beat alternation in electrical and calcium signaling of a cardiac myocyte stimulated at a fixed frequency is well documented to be proarrhythmic. Nonetheless, the involvement of alternans in the processes promoting paroxysmal atrial fibrillation (pAF) is unknown. As such, we set out to ascertain if alternans contribute to the arrhythmogenic mechanisms in pAF. Methods: Simultaneous action potentials (patch-clamp) and [Ca 2+ ] i (Fluo3) were recorded in right atrial myocytes obtained from patients with sinus rhythm (Ctl; n=16) and pAF (n=8). AP and CaT alternans were acquired and analyzed using a custom-written software. Results: AP duration was similar between pAF and Ctl groups over different pacing frequencies, exempting electrical remodeling in pAF. A higher incidence of CaT alternans (46% vs 18%; P =0.08) and AP alternans (82% vs 52%; P <0.05) was observed in myocytes from pAF patients compared with Ctl ( Figure A ). Additionally, the minimum frequency at which AP alternans was detected was significantly lower in pAF (2.4 ± 0.8 vs 4.6 ± 0.6 Hz; n/N=9/6 pAF vs 13/12 Ctl; P <0.05; Figure B ). However, the AP restitution was unchanged in Ctl and pAF myocytes ( Figure C ), thus suggesting CaT alternans as the primary driver of AP alternation ( Figure D ). Conclusion: Atrial myocytes from pAF patients exhibit a higher predisposition to cellular alternans that may alter excitation-propagation in the atria and contribute to the arrhythmogenic substrate in these patients.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used for personalised medicine and preclinical cardiotoxicity testing. Reports on hiPSC-CM commonly describe heterogenous functional readouts and underdeveloped or immature phenotypical properties. Cost-effective, fully defined monolayer culture is approaching mainstream adoption; however, the optimal age at which to utilise hiPSC-CM is unknown. In this study, we identify, track and model the dynamic developmental behaviour of key ionic currents and Ca 2+ -handling properties in hiPSC-CM over long-term culture (30–80 days). hiPSC-CMs > 50 days post differentiation show significantly larger I Ca,L density along with an increased I Ca,L -triggered Ca 2+ -transient. I Na and I K 1 densities significantly increase in late-stage cells, contributing to increased upstroke velocity and reduced action potential duration, respectively. Importantly, our in silico model of hiPSC-CM electrophysiological age dependence confirmed I K 1 as the key ionic determinant of action potential shortening in older cells. We have made this model available through an open source software interface that easily allows users to simulate hiPSC-CM electrophysiology and Ca 2+ -handling and select the appropriate age range for their parameter of interest. This tool, together with the insights from our comprehensive experimental characterisation, could be useful in future optimisation of the culture-to-characterisation pipeline in the field of hiPSC-CM research.

Background: Calcium handling abnormalities contribute to the pathophysiology of atrial fibrillation (AF). In ventricular myocytes, Ca 2+ has been shown to play an important role in mitochondrial response to increased workload by activating key dehydrogenases in the Krebs cycle, necessary for NAD(P)H regeneration and, ultimately, neutralisation. Objective: We hypothesise that workload response of mitochondria is impaired in atrial cardiomyocytes from patients with AF. Methods: Membrane currents (patch clamp), cytosolic Ca 2+ (Fluo-3) and NAD(P)H/FAD autofluorescence were recorded in right atrial myocytes from sinus rhythm (CTL) or AF patients. Immunofluorescence labeling together with STED microscopy and EM microscopy were employed to characterize interaction between mitochondria and sarcoplasmic reticulum (SR). Results: Diastolic [Ca 2+ ] i of voltage-clamped myocytes was comparable, whereas amplitudes of L-type Ca 2+ current and triggered of Ca 2+ transients (CaTs) were decreased by 49% and 43%, respectively. Basal level of NAD(P)H at 0.5 Hz stimulation frequency was comparable in CTL and AF (73.08 6 2.62%, n 5 14/ 7 vs. 81.05 6 5.39%, n 5 4/3 respectively), suggesting a similar redox index. In all CTL cells, upon increasing stimulation frequency to 3 Hz and subsequent b -adrenergic stimulation, the NAD(P)H level initially decreased but recovered to a level comparable to basal state. In contrast, 35% of AF myocytes lost this capacity to recover. Electron and STED microscopy images (Figure) show a disturbance in mitochondrial organisation and their interaction with the SR. Conclusion: Impaired cytosolic Ca 2+ handling and disturbed interaction between mitochondrial and Ca 2+ release sites of the SR may contribute to the impaired redox response of mitochondria to increased workload which we observed in AF patients. This likely results in impaired ATP production and ROS neutralisation, which may finally contribute to atrial arrhythmogenesis in AF patients. ablation coronary sinus (CS) or its branches. Objective: The purpose of this study was to detect predictive value of the new electrocardiographic criterion for differentiating epicardial from endocardial location of posteroseptal accessory pathway. Methods: Seventy-five patients with successful ablation of manifest posteroseptal AP were retrospectively included in our study. Preexcited 12-lead ECGs obtained during sinus rhythm were analyzed. We reviewed the characteristics of the initial 40 msec of the delta wave in lead V1, measured from the earliest QRS deflection in 12 leads. Already described ECG findings suggestive for epicardial location have been considered (negative delta wave in DII, high amplitude S wave in V6). Results: Of 75 patients with posteroseptal AP that undergone catheter ablation, 40 patients (53.3%) had successful epicardial ablation from coronary sinus or its branches. An initial isoelectric or biphasic delta wave in lead V1 proved the highest positive predictive value (97%), sensitivity (82.5 %) and specificity (97 %) for an epicardial location of the posteroseptal AP. Deep S wave in V6 proved lower sensitivity (37.5%) and positive predictive value (68%), but higher specificity (80%) for epicardial location of AP. The specificity and sensitivity for epicardial location of AP of negative delta wave in DII were lower and failed to reach statistical significance. Conclusion: This study shows that an initially isoelectric or biphasic delta wave criterion in lead V1 has a higher specificity, sensitivity and positive predictive value for discrimination of epicardial versus endocardial location of posteroseptal APs than previously described ECG markers. This simple ECG finding could be used to guide the ablationprocedure, reduce its duration and number of failed ablation attempts. alez-Torrecilla Background: Atrioventricular nodal re-entrant tachycardia (AVNRTs) and orthodromic reciprocating tachycardias (ORTs) are major mechanisms of paroxysmal supraventricular tachycardias. Cannon A waves, rapid and regular pulsations with bulging of the internal jugular veins, is traditionally associated with AVNRTs. Objective: The aim was to assess the diagnostic utility of the cannon A wave in cases of paroxysmal supraventricular tachycardias. Methods: We prospectively included 100 patients with paroxysmal supraventricular tachycardias. Videos of the jugular venous pulse were obtained after tachycardia induction. Two independent experts visualized the videos in a blindly manner and classified patients into two groups whether de sign was present or not. Central venous pressure (CVP) was continuously monitored.

Dilated cardiomyopathy (DCM) is a major risk factor for heart failure and is associated with the development of life-threatening cardiac arrhythmias. Using a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model harbouring a mutation in cardiac troponin T (R173W), we aim to examine the cellular basis of arrhythmogenesis in DCM patients with this mutation. iPSC from control (Ctrl) and DCM-TnT-R173W donors from the same family were differentiated into iPSC-CM and analysed through optical action potential (AP) recordings, simultaneous measurement of cytosolic calcium concentration ([Ca 2+ ] i ) and membrane currents and separately assayed using field stimulation to detect the threshold for AP- and [Ca 2+ ] i -alternans development. AP duration was unaltered in TnT-R173W iPSC-CM. Nevertheless, TnT-R173W iPSC-CM showed a strikingly low stimulation threshold for AP- and [Ca 2+ ] i -alternans. Myofilaments are known to play a role as intracellular Ca 2+ buffers and here we show increased Ca 2+ affinity of intracellular buffers in TnT-R173W cells, indicating increased myofilament sensitivity to Ca 2+ . Similarly, EMD57033, a myofilament Ca 2+ sensitiser, replicated the abnormal [Ca 2+ ] i dynamics observed in TnT-R173W samples and lowered the threshold for alternans development. In contrast, application of a Ca 2+ desensitiser (blebbistatin) to TnT-R173W iPSC-CM was able to phenotypically rescue Ca 2+ dynamics, normalising Ca 2+ transient profile and minimising the occurrence of Ca 2+ alternans at physiological frequencies. This finding suggests that increased Ca 2+ buffering likely plays a major arrhythmogenic role in patients with DCM, specifically in those with mutations in cardiac troponin T. In addition, we propose that modulation of myofilament Ca 2+ sensitivity could be an effective anti-arrhythmic target for pharmacological management of this disease.

Dilated cardiomyopathy (DCM) is a major risk factor for heart failure and is associated with the development of life threatening cardiac arrhythmias. It has been shown previously that Ca2+-handling abnormalities may contribute to the pathogenesis of DCM patients harboring a mutation in troponin T (R173W). Whether the altered Ca2+-handling may also contribute to arrhythmogenesis in those patients is currently unknown.

BACKGROUND:Phosphodiesterases (PDE) critically regulate myocardial cAMP and cGMP levels. PDE2 is stimulated by cGMP to hydrolyze cAMP, mediating a negative crosstalk between both pathways. PDE2 upregulation in heart failure contributes to desensitization to β-adrenergic overstimulation. After isoprenaline (ISO) injections, PDE2 overexpressing mice (PDE2 OE) were protected against ventricular arrhythmia. Here, we investigate the mechanisms underlying the effects of PDE2 OE on susceptibility to arrhythmias.METHODS:Cellular arrhythmia, ion currents, and Ca2+-sparks were assessed in ventricular cardiomyocytes from PDE2 OE and WT littermates.RESULTS:Under basal conditions, action potential (AP) morphology were similar in PDE2 OE and WT. ISO stimulation significantly increased the incidence of afterdepolarizations and spontaneous APs in WT, which was markedly reduced in PDE2 OE. The ISO-induced increase in ICaL seen in WT was prevented in PDE2 OE. Moreover, the ISO-induced, Epac- and CaMKII-dependent increase in INaL and Ca2+-spark frequency was blunted in PDE2 OE, while the effect of direct Epac activation was similar in both groups. Finally, PDE2 inhibition facilitated arrhythmic events in ex vivo perfused WT hearts after reperfusion injury.CONCLUSION:Higher PDE2 abundance protects against ISO-induced cardiac arrhythmia by preventing the Epac- and CaMKII-mediated increases of cellular triggers. Thus, activating myocardial PDE2 may represent a novel intracellular anti-arrhythmic therapeutic strategy in HF.

Aims Atrial fibrillation (AF) is a commonly occurring arrhythmia after cardiac surgery (postoperative AF, poAF) and is associated with poorer outcomes. Considering that reduced atrial contractile function is a predictor of poAF and that Ca2+ plays an important role in both excitation-contraction coupling and atrial arrhythmogenesis, this study aims to test whether alterations of intracellular Ca2+ handling contribute to impaired atrial contractility and to the arrhythmogenic substrate predisposing patients to poAF. Methods and results Right atrial appendages were obtained from patients in sinus rhythm undergoing open-heart surgery. Cardiomyocytes were investigated by simultaneous measurement of [Ca2+], and action potentials (APs, patch-clamp). Patients were followed-up for 6 days to identify those with and without poAF. Speckle-tracking analysis of preoperative echocardiography revealed reduced left atrial contraction strain in poAF patients. At the time of surgery, cellular Ca2+ transients (CaTs) and the sarcoplasmic reticulum (SR) Ca2+ content were smaller in the poAF group. CaT decay was slower in poAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting preserved sodium-calcium exchanger function. In agreement, western blots revealed reduced SERCA2a expression in poAF patients but unaltered phosphotamban expression/phosphorylation. Computational modelling indicated that reduced SERCA activity promotes occurrence of CaT and AP alternans. Indeed, alternans of CaT and AP occurred more often and at lower stimulation frequencies in atrial myocytes from poAF patients. Resting membrane potential and AP duration were comparable between both groups at various pacing frequencies (0.25-8 Hz). Conclusion Biochemical, functional, and modelling data implicate reduced SERCA-mediated Ca2+ reuptake into the SR as a major contributor to impaired preoperative atriat contractile function and to the pre-existing arrhythmogenic substrate in patients developing poAF. [GRAPHICS] .

In this study, we use patient-specific induced pluripotent stem cells (ps-iPSCs) to gain insights into Tetralogy of Fallot (TOF), which represents the most common cyanotic heart defect in humans. Patient-specific expression patterns and genetic variability were investigated in iPSCs and derived cardiomyocytes (CMs) using whole genome and transcriptome sequencing data. First, the clonal mutational burden of the iPSCs was studied, which revealed in two out of three iPSC lines of one patient a somatic mutation in the DNA-binding domain of tumor suppressor P53 that was not observed in the genomic DNA from blood. Characterization of this mutation showed its functional impact, which makes the cells inappropriate for modelling and studying the disease. For the other patient, potential disease-relevant differential gene expression between and across cardiac differentiation was shown. Here, clear differences at the later stages of differentiation could be observed. In addition, there were abnormalities in the patient-specific CMs based on intracellular calcium handling, cell contraction and action potential dynamics. Overall, this study provides first insights into the complex molecular and functional mechanisms underlying iPSC-derived cardiomyocyte differentiation and its alterations in TOF, which might also have an impact on the long-term clinical outcome and management of these patients.

To support scientific exchange and activity in the field of cardiac cellular electrophysiology, the German Cardiac Society Working Group on Cellular Electrophysiology (AG 18) established a two-day symposium to be held every 2 years. The second Ion Channel Symposium entitled “Göttingen Channels 2017—Of Benches and Beds” took place in Göttingen from September 22nd to 23rd under the auspices of the German Cardiac Society. A group of national and international experts presented scientific advances in cardiac electrophysiology and rhythmology. The symposium’s primary focus was the significance of cellular electrophysiology findings for the optimization of diagnostic and therapeutic strategies against cardiac arrhythmias. To this end, speakers, chairpersons and attendees discussed the contribution of specific molecular alterations to the initiation and perpetuation of atrial and ventricular arrhythmias. Furthermore, the meeting highlighted how discoveries in electrophysiological research may lead to novel therapeutic targets. The interdisciplinary assessment of mechanisms and therapeutic strategies of cardiac arrhythmias represented a key feature of the meeting. A unique combination of topics and speakers representing both basic science and clinical electrophysiology ensured the scientific success of the “Göttingen Channels 2017” symposium. The next Ion Channel Symposium is planned to be hosted by the incoming co-chair of the German Cardiac Society Working Group on Cellular Electrophysiology in fall 2019.

Rationale: Phosphodiesterase 2 is a dual substrate esterase, which has the unique property to be stimulated by cGMP, but primarily hydrolyzes cAMP. Myocardial phosphodiesterase 2 is upregulated in human heart failure, but its role in the heart is unknown.Objective: To explore the role of phosphodiesterase 2 in cardiac function, propensity to arrhythmia, and myocardial infarction.Methods and Results: Pharmacological inhibition of phosphodiesterase 2 (BAY 60-7550, BAY) led to a significant positive chronotropic effect on top of maximal beta-adrenoceptor activation in healthy mice. Under pathological conditions induced by chronic catecholamine infusions, BAY reversed both the attenuated beta-adrenoceptor-mediated inotropy and chronotropy. Conversely, ECG telemetry in heart-specific phosphodiesterase 2-transgenic (TG) mice showed a marked reduction in resting and in maximal heart rate, whereas cardiac output was completely preserved because of greater cardiac contraction. This well-tolerated phenotype persisted in elderly TG with no indications of cardiac pathology or premature death. During arrhythmia provocation induced by catecholamine injections, TG animals were resistant to triggered ventricular arrhythmias. Accordingly, Ca2+-spark analysis in isolated TG cardiomyocytes revealed remarkably reduced Ca2+ leakage and lower basal phosphorylation levels of Ca2+-cycling proteins including ryanodine receptor type 2. Moreover, TG demonstrated improved cardiac function after myocardial infarction.Conclusions: Endogenous phosphodiesterase 2 contributes to heart rate regulation. Greater phosphodiesterase 2 abundance protects against arrhythmias and improves contraction force after severe ischemic insult. Activating myocardial phosphodiesterase 2 may, thus, represent a novel intracellular antiadrenergic therapeutic strategy protecting the heart from arrhythmia and contractile dysfunction.

The purpose of this article is to review the basis of arrhythmogenesis, the functional and clinical role of the late Na current, and its therapeutic inhibition. Under pathological conditions such as ischemia and heart failure this current is abnormally enhanced and influences cellular electrophysiology as a proarrhythmic substrate in myocardial pathology. Ranolazine the only approved late Na current blocker has been demonstrated to produce antiarrhythmic effects in the atria and the ventricle. We summarize recent experimental and clinical studies of ranolazine and other experimental late Na current blockers and discuss the significance of the available data.

Phosphodiesterase 2 (PDE2) is a dual substrate enzyme, hydrolyzing both cAMP and cGMP. We showed that myocardial PDE2 is upregulated in human and experimental heart failure (HF) while PDE3 and PDE4 are reduced. To explore the pathophysiological consequences of enhanced PDE2 activity, transgenic mice with a heart-specific overexpression of the PDE2A3 isoform (PDE2-TG) were generated. PDE2 activity was measuredin heart extractsby radioenzymatic assay. Sarcomere shortening, Ca2+ transients and L-type Ca2+ current (ICa,L) were recorded in adult ventricular cardiomyocytes from wild-type (WT) and PDE2-TG mice. SR Ca2+ leak was estimated with tetracaine (RyR blocker) and spontaneous Ca2+ waves (SCW) were recorded during 30s pacing pause. Intracellular cAMP level was measured with FRET. Phosphorylation level of excitation-contraction coupling (ECC) proteins was determined by Western Blot. Heart function was investigated by echocardiography and ECG-telemetry. Isoprenaline (ISO) was used to compare β-adrenergic (β-AR) response of all parameters in WT and PDE2-TG mice. cAMP and cGMP-PDE2 activity was strongly increased in PDE2-TG as compared to WT mice. Resting and maximal heart rate was markedly reduced in PDE2-TG mice. The β-AR stimulation of cell contractility, Ca2+ transient, ICa,L and [cAMP]i was severely blunted. PDE2-TG mice showed reduced SR Ca2+ leak and SCW (both at the cellular and in vivo levels), without changes in Ca2+ load as compared to WT during β-AR activation. PDE2-TG mice showed a lower basal phosphorylation of ECC proteins and have a longer lifespan than their WT littermates. All effects of PDE2 overexpression were reversed by PDE2 inhibitor, Bay-607550. Our results demonstrate that PDE2 plays a critical role in the regulation of cardiac ECC. PDE2 overexpression appears to protect the cardiomyocytes by reducing Ca2+-leakage and arrhythmias during β-AR stimulation. PDE2 may represent a new therapeutic strategy in heart failure.

INTRODUCTION:Pharmacological rhythm control of atrial fibrillation (AF) in patients with structural heart disease is limited. Ranolazine in combination with low dose dronedarone remarkably reduced AF-burden in the phase II HARMONY trial. We thus aimed to investigate the possible mechanisms underlying these results.METHODS AND RESULTS:Patch clamp experiments revealed that ranolazine (5μM), low-dose dronedarone (0.3μM), and the combination significantly prolonged action potential duration (APD90) in atrial myocytes from patients in sinus rhythm (prolongation by 23.5±0.1%, 31.7±0.1% and 25.6±0.1% respectively). Most importantly, in atrial myocytes from patients with AF ranolazine alone, but more the combination with dronedarone, also prolonged the typically abbreviated APD90 (prolongation by 21.6±0.1% and 31.9±0.1% respectively). It was clearly observed that neither ranolazine, dronedarone nor the combination significantly changed the APD or contractility and twitch force in ventricular myocytes or trabeculae from patients with heart failure (HF). Interestingly ranolazine, and more so the combination, but not dronedarone alone, caused hyperpolarization of the resting membrane potential in cardiomyocytes from AF. As measured by confocal microscopy (Fluo-3), ranolazine, dronedarone and the combination significantly suppressed diastolic sarcoplasmic reticulum (SR) Ca(2+) leak in myocytes from sinus rhythm (reduction by ranolazine: 89.0±30.7%, dronedarone: 75.6±27.4% and combination: 78.0±27.2%), in myocytes from AF (reduction by ranolazine: 67.6±33.7%, dronedarone: 86.5±31.7% and combination: 81.0±33.3%), as well as in myocytes from HF (reduction by ranolazine: 64.8±26.5% and dronedarone: 65.9±29.3%).CONCLUSIONS:Electrophysiological measurements during exposure to ranolazine alone or in combination with low-dose dronedarone showed APD prolongation, cellular hyperpolarization and reduced SR Ca(2+) leak in human atrial myocytes. The combined inhibitory effects on various currents, in particular Na(+) and K(+) currents, may explain the anti-AF effects observed in the HARMONY trial. Therefore, the combination of ranolazine and dronedarone, but also ranolazine alone, may be promising new treatment options for AF, especially in patients with HF, and merit further clinical investigation.