Search

Aim: In healthy hearts, ventricular gap junctions are mainly composed by connexin43 (Cx43) and localize in the intercalated disc, enabling appropriate electrical coupling. In diseased hearts, Cx43 is heterogeneously down-regulated, whereas activity of calmodulin/calcium-calmodulin protein kinase II (CaM/CaMKII) signalling increases. It is unclear if CaM/CaMKII affects Cx43 expression/localization or impulse propagation. We analysed different models to assess this. Methods and results: AC3-I mice with CaMKII genetically inhibited were subjected to pressure overload (16 weeks, TAC vs. sham). Optical and epicardial mapping was performed on Langendorff-perfused rabbit and AC3-I hearts, respectively. Cx43 subcellular distribution from rabbit/mouse ventricles was evaluated by immunoblot after Triton X-100-based fractionation. In mice with constitutively reduced CaMKII activity (AC3-I), conduction velocity (CV) was augmented (n = 11, P, 0.01 vs. WT); in AC3-I, CV was preserved after TAC, in contrast to a reduction seen in TAC-WT mice (220%). Cx43 expression was preserved after TAC in AC3-I mice, though arrhythmias and fibrosis were still present. In rabbits, W7 (CaM inhibitor, 10 mM) increased CV (6-13%, n = 6, P, 0.05), while susceptibility to arrhythmias decreased. Immunoconfocal microscopy revealed enlarged Cx43 cluster sizes at intercalated discs of those hearts. Total Cx43 did not change by W7 (n = 4), whereas Triton X-100 insoluble Cx43 increased (+21%, n = 4, P, 0.01). Similar findings were obtained in AC3-I mouse hearts when compared with control, and in cultured dog cardiomyocytes. Functional implication was shown through increased intercellular coupling in cultured neonatal rat cardiomyocytes. Conclusion: Both acute and chronic CaM/CaMKII inhibition improves conduction characteristics and enhances localization of Cx43 in the intercalated disc. In the absence of fibrosis, this reduced the susceptibility for arrhythmias. [ABSTRACT FROM AUTHOR]

Aims: Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is caused by mutations in the cardiac ryanodine receptor (RyR2) that lead to disrupted Ca2+ handling in cardiomyocytes and ventricular tachycardia. The aim of this study was to test whether exercise training could reduce the propensity for arrhythmias in mice with the CPVT1-causative missense mutation Ryr2-R2474S by restoring normal Ca2+ handling. Methods and results:Ryr2-R2474S mice (RyR-RS) performed a 2 week interval treadmill exercise training protocol. Each exercise session comprised five 8 min intervals at 80-90% of the running speed at maximal oxygen uptake (VO2max) and 2 min active rest periods at 60%. VO2max increased by 10±2% in exercise trained RyR-RS (ET), while no changes were found in sedentary controls (SED). RyR-RS ET showed fewer episodes of ventricular tachycardia compared with RyR-RS SED, coinciding with fewer Ca2+ sparks and waves, less diastolic Ca2+ leak from the sarcoplasmic reticulum, and lower phosphorylation levels at RyR2 sites associated with Ca2+-calmodulin-dependent kinase type II (CaMKII) compared with RyR-RS SED. The CaMKII inhibitor autocamtide-2-related inhibitory peptide and also the antioxidant N-acetyl-L-cysteine reduced Ca2+ wave frequency in RyR-RS equally to exercise training. Protein analysis as well as functional data indicated a mechanism depending on reduced levels of oxidized CaMKII after exercise training. Two weeks of detraining reversed the beneficial effects of the interval treadmill exercise training protocol in RyR-RS ET. Conclusion: Long-term effects of interval treadmill exercise training reduce ventricular tachycardia episodes in mice with a CPVT1- causative Ryr2 mutation through lower CaMKII-dependent phosphorylation of RyR2. [ABSTRACT FROM AUTHOR]

Aims Most cardiac arrhythmia-associated genes encode ion channel subunits and regulatory proteins that are also expressed outside the heart, suggesting that diseases linked to their disruption may be multifactorial. KCNE2 is a ubiquitously expressed potassium channel b subunit associated with cardiac arrhythmia, atherosclerosis, and myocardial infarction (MI) in human populations. Here, we tested the hypothesis that Kcne2 disruption in mice would influence the acute outcome of experimentally induced MI. Methods and results One-year-old male Kcne2+/+ and Kcne2-/- mice were subjected to cardiac ischaemia/reperfusion injury (IRI) by left anterior descending coronary artery ligation. After reperfusion (3 h), infarct size and markers of tissue damage were quantified. Unexpectedly, post-reperfusion, Kcne2-/- mice exhibited 40% lower infarct size, decreased myocardial apoptosis and damage, and more than two-fold lower serum levels of damage markers, lactate dehydrogenase and creatine kinase, than Kcne2+/+ mice. Kcne2 deletion, despite increasing normalized heart weight and prolonging baseline QTc by 70%, helped preserve post-infarct cardiac function (quantified by a Millar catheter), with parameters including left ventricular maximum pressure, max dP/dt (P < 0.01), contractility index, and pressure/time index (P < 0.05) all greater in Kcne2-/- compared with Kcne2+/+ mice. Western blotting indicated two-fold-increased glycogen synthase kinase 3b (GSK-3b) phosphorylation (inactivation) before and after IRI (P < 0.05) in Kcne2-/- mice compared with Kcne2+/+ mice. GSK-3b inhibition by SB216763 mimicked in Kcne2+/+ mice the cardioprotective effects of Kcne2 deletion, but did not further enhance them in Kcne2-/- mice, suggesting that GSK-3b inactivation was a primary cardioprotective mechanism arising from Kcne2 deletion. Conclusions Kcne2 deletion preconditions the heart, attenuating the acute tissue damage caused by an imposed IRI. The findings contribute further evidence that genetic disruption of arrhythmia-associated ion channel genes has cardiac ramifications beyond abnormal electrical activity. [ABSTRACT FROM AUTHOR]

Aims: A major concern of using phosphodiesterase (PDE) inhibitors in heart failure is their potential to increase mortality by inducing arrhythmias. By diminishing cyclic adenosine monophosphate (cAMP) hydrolysis, they promote protein kinase A (PKA) activity under b-adrenergic receptor (b-AR) stimulation, hence enhancing Ca2+ cycling and contraction. Yet, cAMP also activates CaMKII via PKA or the exchange protein Epac, but it remains unknown whether these pathways are involved in the pro-arrhythmic effect of PDE inhibitors. Methods and results: Excitation-contraction coupling was investigated in isolated adult rat ventricular myocytes loaded with Fura-2 and paced at 1 Hz allowing coincident measurement of intracellular Ca2+ and sarcomere shortening. The PDE4 inhibitor Ro 20-1724 (Ro) promoted the inotropic effects of the non-selective β-AR agonist isoprenaline (Iso) and also spontaneous diastolic Ca2+ waves (SCWs). PDE4 inhibition potentiated RyR2 and PLB phosphorylation at specific PKA and CaMKII sites increasing sarcoplasmic reticulum (SR) Ca2+ load and SR Ca2+ leak measured in a 0Na+/0Ca2+ solution±tetracaine. PKA inhibition suppressed all the effects of Iso ± Ro, whereas CaMKII inhibition prevented SR Ca2+ leak and diminished SCW incidence without affecting the inotropic effects of Ro. Inhibition of Epac2 but not Epac1 diminished the occurrence of SCWs. PDE3 inhibition with cilostamide induced an SR Ca2+ leak, which was also blocked by CaMKII inhibition. Conclusion Our results show that PDE inhibitors exert inotropic effects via PKA but lead to SCWs via both PKA and CaMKII activation partly via Epac2, suggesting the potential use of CaMKII inhibitors as adjuncts to PDE inhibition to limit their pro-arrhythmic effects. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is an extremely common clinical problem associated with increased morbidity and mortality. Current antiarrhythmic options include pharmacological, ablation, and surgical therapies, and have significantly improved clinical outcomes. However, their efficacy remains suboptimal, and their use is limited by a variety of potentially serious adverse effects. There is a clear need for improved therapeutic options. Several decades of research have substantially expanded our understanding of the basic mechanisms of AF. Ectopic firing and re-entrant activity have been identified as the predominant mechanisms for arrhythmia initiation and maintenance. However, it has become clear that the clinical factors predisposing to AF and the cellular and molecular mechanisms involved are extremely complex. Moreover, all AF-promoting and maintaining mechanisms are dynamically regulated and subject to remodelling caused by both AF and cardiovascular disease. Accordingly, the initial presentation and clinical progression of AF patients are enormously heterogeneous. An understanding of arrhythmia mechanisms is widely assumed to be the basis of therapeutic innovation, but while this assumption seems self-evident, we are not aware of any papers that have critically examined the practical contributions of basic research into AFmechanisms to arrhythmia management. Here, we review recent insights into the basic mechanisms of AF, critically analyse the role of basic research insights in the development of presently used anti-AF therapeutic options and assess the potential value of contemporary experimental discoveries for future therapeutic innovation. Finally, we highlight some of the important challenges to the translation of basic science findings to clinical application. [ABSTRACT FROM AUTHOR]

Although the initiation, development, and maintenance of atrial fibrillation (AF) have been linked to alterations in myocyte redox state, the field lacks a complete understanding of the impact these changes may have on cellular signalling, atrial electrophysiology, and disease progression. Recent studies demonstrate spatiotemporal changes in reactive oxygen species production shortly after the induction of AF in animal models with an uncoupling of nitric oxide synthase activity ensuing in the presence of long-standing persistent AF, ultimately leading to a major shift in nitroso-redox balance. However, it remains unclear which radical or non-radical species are primarily involved in the underlying mechanisms of AF or which proteins are targeted for redox modification. In most instances, only free radical oxygen species have been assessed; yet evidence from the redox signalling field suggests that non-radical species are more likely to regulate cellular processes. A wider appreciation for the distinction of these species and how both species may be involved in the development and maintenance of AF could impact treatment strategies. In this review, we summarize how redox second-messenger systems are regulated and discuss the recent evidence for alterations in redox regulation in the atrial myocardium in the presence of AF, while identifying some critical missing links. We also examine studies looking at antioxidants for the prevention and treatment of AF and propose alternative redox targets that may serve as superior therapeutic options for the treatment of AF. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common cardiac arrhythmia with well-established clinical and genetic risk components. Genome-wide association studies (GWAS) have identified 17 independent susceptibility signals for AF at 14 genomic regions, but the mechanisms through which these loci confer risk to AF remain largely undefined. This problem is not unique to AF, as the field of functional genomics, which attempts to bridge this gap from genotype to phenotype, has only uncovered the mechanisms for a handful ofGWAS loci. Recent functional genomic studies have made great strides towards translating genetic discoveries to an underlying mechanism, but the large-scale application of these techniques to AF has remain limited. These advances, as well as the continued unresolved challenges for both common variation in AF and the functional genomics field in general, will be the subject of the following review. [ABSTRACT FROM AUTHOR]

Aims Atrial fibrillation (AF) is a common arrhythmia with an important heritable aspect. The genetic factors underlying AF have not been fully elucidated. Methods and results We screened six candidate genes (CAV1, KCNJ2, KCNQ1, NKX2.5, PITX2, and TBX5) for novel mutations in 139 patients of Chinese descent with early-onset AF and 576 controls. Four missense TBX5 mutations, p.R355C, p.Q376R, p.A428S, and p.S372L, were identified in evolutionarily conserved regions. We did not find any mutations in CAV1, KCNJ2, KCNQ1, NKX2.5, and PITX2. These mutations increased the expression of atrial natriuretic peptide (ANP) and connexin-40 (CX40) in the primarily cultured rat atrial myocytes but did not alter the expression of cardiac structural genes, atrial myosin heavy chain-α (MHC-α) and myosin lightchain-2α (MLC-2α). Overexpression of p.R355C developed an atrial arrhythmia suggestive of paroxysmal AF in the zebrafish model. To replicate our findings, we screened TBX5 in 527 early-onset AF cases from the Massachusetts General Hospital AF study. A novel TBX5 deletion (ΔAsp118, p.D118del) was identified, while no TBX5 mutations were identified in 1176 control subjects. Conclusion Our results provide both genetic and functional evidence to support the contribution of TBX5 gene in the pathogenesis of AF. The potential mechanism of arrhythmia may be due in part to the disturbed expression of ANP and CX40. [ABSTRACT FROM AUTHOR]

Aims The progression of pathological left ventricular remodelling leads to cardiac dysfunction and contributes to the occurrence of malignant arrhythmias and sudden cardiac death. The underlying molecular mechanisms remain unclear, however. Our aim was to examine the role of the renin–angiotensin system (RAS) in the mechanism underlying arrhythmogenic cardiac remodelling using a transgenic mouse expressing a cardiac-specific dominant-negative form of neuron-restrictive silencer factor (dnNRSF-Tg). This mouse model exhibits progressive cardiac dysfunction leading to lethal arrhythmias. Methods and results Subcutaneous administration of aliskiren, a direct renin inhibitor, significantly suppressed the progression of pathological cardiac remodelling and improved survival among dnNRSF-Tg mice while reducing arrhythmogenicity. Genetic deletion of the angiotensin type 1a receptor (AT1aR) similarly suppressed cardiac remodelling and sudden death. In optical mapping analyses, spontaneous ventricular tachycardia (VT) and fibrillation (VF) initiated by breakthrough-type excitations originating from focal activation sites and maintained by functional re-entry were observed in dnNRSF-Tg hearts. Under constant pacing, dnNRSF-Tg hearts exhibited markedly slowed conduction velocity, which likely contributes to the arrhythmogenic substrate. Aliskiren treatment increased conduction velocity and reduced the incidence of sustained VT. These effects were associated with suppression of cardiac fibrosis and restoration of connexin 43 expression in dnNRSF-Tg ventricles. Conclusion Renin inhibition or genetic deletion of AT1aR suppresses pathological cardiac remodelling that leads to the generation of substrates maintaining VT/VF and reduces the occurrence of sudden death in dnNRSF-Tg mice. These findings demonstrate the significant contribution of RAS activation to the progression of arrhythmogenic substrates. [ABSTRACT FROM AUTHOR]

Aims: In atrial fibrillation (AF), abnormalities in Ca2+ release contribute to arrhythmia generation and contractile dysfunction. We explore whether RyR cluster ultrastructure is altered and is associated with functional abnormalities in AF. Methods and Results: Using high resolution confocal microscopy (STED) we examined RyR cluster morphology in fixed atrial myocytes from sheep with persistent AF (N=6) and control (Ctrl; N=6) animals. RyR clusters on average contained 15 contiguous RyRs; this did not differ between AF and Ctrl. However, the distance between clusters was significantly reduced in AF (288±12 nm vs. 376±17 nm). When RyR clusters were grouped into Ca2+ release units (CRUs), i.e. clusters separated by <150 nm), CRUs in AF had more clusters (3.43±0.10 vs. 2.95±0.02 in Ctrl), which were more dispersed. Furthermore, in AF cells, more RyR clusters were found between Z lines. In parallel experiments, Ca2+ sparks were monitored in live permeabilized myocytes. In AF, myocytes had: (i) >50% higher spark frequency with (ii) increased spark time-to-peak (TTP) and duration and (iii) a higher incidence of macrosparks. A computational model of the CRU was used to simulate the morphological alterations observed in AF cells. Increasing cluster fragmentation to the level observed in AF cells caused the observed changes i.e. higher spark frequency, increased TTP and duration; RyR clusters dispersed between Z-lines increased the occurrence of macrosparks. Conclusion: In persistent AF, ultrastructural reorganization of RyR clusters within CRUs is associated with overactive Ca2+ release, increasing the likelihood of propagating Ca2+ release. [ABSTRACT FROM AUTHOR]

Aims: Cardiac calsequestrin (CASQ2) and histidine-rich Ca-binding protein (HRC) are sarcoplasmic reticulum (SR) Ca-binding proteins that regulate SR Ca release in mammalian heart. Deletion of either CASQ2 or HRC results in relatively mild phenotypes characterized by preserved cardiac structure and function, although CASQ2 knockout (KO), or Cnull, shows increased arrhythmia burden under conditions of catecholaminergic stress. We hypothesized that given the apparent overlap of functions of CASQ2 and HRC, simultaneous ablation of both would deteriorate the cardiac phenotype compared with the single knockouts. Methods and results: In contrast to this expectation, double knockout (DKO) mice lacking both CASQ2 and HRC exhibited normal cardiac ejection fraction and ultrastructure. Moreover, the predisposition to catecholamine-dependent arrhythmia that characterizes the Cnull phenotype was alleviated in the DKOmice. At the myocyte level, DKOmice displayed Ca transients of normal amplitude; additionally, the frequency of spontaneous Ca waves and sparks in the presence of isoproterenol were decreased markedly compared with Cnull. Furthermore, restitution of SR Ca release was slowed in DKO myocytes compared with Cnull cells. Conclusion: Our results suggest that rather than being functionally redundant, CASQ2 and HRC modulate cardiac ryanodine receptor- mediated (RyR2) Ca release in an opposing manner. In particular, while CASQ2 stabilizes RyR2 rendering it refractory in the diastolic phase, HRC enhances RyR2 activity facilitating RyR2 recovery from refractoriness. [ABSTRACT FROM AUTHOR]

Aims: Premature ventricular complexes (PVCs) due to spontaneous calcium (Ca) release (SCR) events at the cell level can precipitate ventricular arrhythmias. However, the mechanistic link between SCRs and PVC formation remains incompletely understood. The aim of this study was to investigate the conditions under which delayed afterdepolarizations resulting from stochastic subcellular SCR events can overcome electrotonic source-sink mismatch, leading to PVC initiation. Methods and results: A stochastic subcellular-scale mathematical model of SCR was incorporated in a realistic model of the rabbit ventricles and Purkinje system (PS). Elevated levels of diastolic sarcoplasmic reticulum Ca2+ (CaSR) were imposed until triggered activity was observed, allowing us to compile statistics on probability, timing, and location of PVCs. At CaSR= 1500 µmol/L PVCs originated in the PS. When SCR was incapacitated in the PS, PVCs also emerged in the ventricles, but at a higher CaSR (=1550 µmol/L) and with longer waiting times. For each model configuration tested, the probability of PVC occurrence increased from 0 to 100% within a well-defined critical CaSR range; this transition was much more abrupt in organ-scale models (~50 µmol/L CaSR range) than in the tissue strand (~100 µmol/L) or single-cell (~450 µmol/L) models. Among PVCs originating in the PS, ~68% were located near Purkinje-ventricular junctions (<1 mm). Conclusion: SCR events overcome source-sink mismatch to trigger PVCs at a critical CaSR threshold. Above this threshold, PVCs emerge due to increased probability and reduced variability in timing of SCR events, leading to significant diastolic depolarization. Sites of lower electronic load, such as the PS, are preferential locations for triggering. [ABSTRACT FROM AUTHOR]

Aim TRPC3 is a non-selective cation channel, which forms a Ca2+ entry pathway involved in cardiac remodelling. Our aim was to analyse acute electrophysiological and contractile consequences of TRPC3 activation in the heart. Methods and results We used a murine model of cardiac TRPC3 overexpression and a novel TRPC3 agonist, GSK1702934A, to uncover (patho)physiological functions of TRPC3. GSK1702934A induced a transient, non-selective conductance and prolonged action potentials in TRPC3-overexpressing myocytes but lacked significant electrophysiological effects in wild-type myocytes. GSK1702934A transiently enhanced contractility and evoked arrhythmias in isolated Langendorff hearts from TRPC3-overexpressing but not wild-type mice. Interestingly, pro-arrhythmic effects outlasted TRPC3 current activation, were prevented by enhanced intracellular Ca2+ buffering, and suppressed by the NCX inhibitor 3',4'-dichlorobenzamil hydrochloride. GSK1702934A substantially promoted NCX currents in TRPC3-overexpressing myocytes. The TRPC3-dependent electrophysiologic, pro-arrhythmic, and inotropic actions of GSK1702934A were mimicked by angiotensin II (AngII). Immunocytochemistry demonstrated colocalization of TRPC3 with NCX1 and disruption of local interaction upon channel activation by either GSK1702934A or AngII. Conclusion Cardiac TRPC3 mediates Ca2+ and Na+ entry in proximity of NCX1, thereby elevating cellular Ca2+ levels and contractility. Excessive activation of TRPC3 is associated with transient cellular Ca2+overload, spatial uncoupling between TRPC3 and NCX1, and arrhythmogenesis. We propose TRPC3-NCX micro/nanodomain communication as determinant of cardiac contractility and susceptibility to arrhythmogenic stimuli. [ABSTRACT FROM AUTHOR]

Aims Duchenne muscular dystrophy (DMD), a degenerative pathology of skeletal muscle, also induces cardiac failure and arrhythmias due to a mutation leading to the lack of the protein dystrophin. In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The absence of dystrophin results in an elevated stress-induced Ca2+ influx due to the inadequate functioning of several proteins, such as stretch-activated channels (SACs). Our aim was to investigate whether transient receptor potential vanilloid channels type 2 (TRPV2) form subunits of the dysregulated SACs in cardiac dystrophy. Methods and results We defined the role of TRPV2 channels in the abnormal Ca2+ influx of cardiomyocytes isolated from dystrophic mdx mice, an established animal model for DMD. In dystrophic cells, western blotting showed that TRPV2 was two-fold over expressed. While normally localized intracellularly, in myocytes from mdx mice TRPV2 channels were translocated to the sarcolemma and were prominent along the T-tubules, as indicated by immunocytochemistry. Membrane localization was confirmed by biotinylation assays. Furthermore, in mdx myocytes pharmacological modulators suggested an abnormal activity of TRPV2, which has a unique pharmacological profile among TRP channels. Confocal imaging showed that these compounds protected the cells from stress-induced abnormal Ca2+ signals. The involvement of TRPV2 in these signals was confirmed by specific pore-blocking antibodies and by small-interfering RNA ablation of TRPV2. Conclusion Together, these results establish the involvement of TRPV2 in a stretch-activated calcium influx pathway in dystrophic cardiomyopathy, contributing to the defective cellular Ca2+ handling in this disease. [ABSTRACT FROM AUTHOR]

Aims Sudden death resulting from cardiac arrhythmias is the most common consequence of cardiac disease. Certain arrhythmias caused byabnormal impulse formation including catecholaminergic polymorphic ventricular tachycardia (CPVT) are associated with delayed afterdepolarizations resulting from diastolic Ca2+ release (DCR) from the sarcoplasmic reticulum (SR). Despite high response of CPVT to agents directly affecting Ca2+ cycling, the incidence of refractory cases is still significant. Surprisingly, these patients often respond to treatment with Na+ channel blockers.However, the relationship between Na+ influx and disturbances in Ca2+ handling immediately preceding arrhythmias in CPVT remains poorly understood and is the object of this study. Methods and results We performed optical Ca2+andmembrane potential imaging in ventricularmyocytes and intact cardiac muscles as well as surface ECGs on a CPVT mouse model with a mutation in cardiac calsequestrin. We demonstrate that a subpopulation of Na+ channels (neuronal Na+ channels; n Nav) colocalize with ryanodine receptor Ca2+ release channels (RyR2). Disruption of the crosstalk between nNav and RyR2 by nNav blockade with riluzole reduced and also desynchronized DCR in isolated cardiomyocytes and in intact cardiac tissue. Such desynchronization of DCR on cellular and tissue level translated into decreased arrhythmias in CPVT mice. Conclusions Thus, our study offers the first evidence that nNav contribute to arrhythmogenic DCR, thereby providing a conceptual basis for mechanism-based antiarrhythmic therapy. [ABSTRACT FROM AUTHOR]

Aims Release of norepinephrine (NE) from sympathetic neurons enhances heart rate (HR) and developed force through activation of β-adrenergic receptors, and this sympathoexcitation is a key risk for the generation of cardiac arrhythmias. Studies of β-adrenergic modulation of cardiac function typically involve the administration of exogenous β-adrenergic receptor agonists to directly elicit global β-adrenergic receptor activation by bypassing the involvement of sympathetic nerve terminals. In this work, we use a novel method to activate sympathetic fibres within the myocardium of Langen-dorff-perfused hearts while measuring changes in electrical and mechanical function. Methods and results The light-activated optogenetic protein channelrhodopsin-2 (ChR2) was expressed in murine catecholaminergic sympathetic neurons. Sympathetic fibres were then photoactivated to examine changes in contractile force, HR, and cardiac electrical activity. Incidence of arrhythmia was measured with and without exposure to photoactivation of sympathetic fibres, and hearts were optically mapped to detect changes in action potential durations and conduction velocities. Results demonstrate facilitation of both developed force and HR after photostimulated release of NE, with increases in contractile force and HR of 34.5 ± 5.5 and 25.0 ± 9.3%, respectively. Photostimulation of sympathetic fibres also made hearts more susceptible to arrhythmia, with greater incidence and severity. In addition, optically mapped action potentials displayed a small but significant shortening of the plateau phase (-5.5 ± 1.0 ms) after photostimulation. Conclusion This study characterizes a powerful and clinically relevant new model for studies of cardiac arrhythmias generated by increasing the activity of sympathetic nerve terminals and the resulting activation of myocyte β-adrenergic receptors. [ABSTRACT FROM AUTHOR]

Aims Myofibroblasts (MFBs) as appearing in the myocardium during fibrotic remodelling induce slow conduction following heterocellular gap junctional coupling with cardiomyocytes (CMCs) in bioengineered tissue preparations kept under isometric conditions. In this study, we investigated the hypothesis that strain as developed during diastolic filling of the heart chambers may modulate MFB-dependent slow conduction. Methods and results Effects of defined levels of strain on single-cell electrophysiology (patch clamp) and impulse conduction in patterned growth cell strands (optical mapping) were investigated in neonatal rat ventricular cell cultures (Wistar) grown on flexible substrates. While 10.5% strain only minimally affected conduction times in control CMC strands (+3.2%, n.s.), it caused a significant slowing of conduction in the fibrosis model consisting of CMC strands coated with MFBs (conduction times +26.3%). Increased sensitivity to strain of the fibrosis model was due to activation of mechanosensitive channels (MSCs) in both CMCs and MFBs that aggravated the MFB-dependent baseline depolarization of CMCs. As found in non-strained preparations, baseline depolarization of CMCs was partly due to the presence of constitutively active MSCs in coupled MFBs. Constitutive activity of MSCs was not dependent on the contractile state of MFBs, because neither stimulation (thrombin) nor suppression (blebbistatin) thereof significantly affected conduction velocities in the non-strained fibrosis model. Conclusions The findings demonstrate that both constitutive and strain-induced activity of MSCs in MFBs significantly enhance their depolarizing effect on electrotonically coupled CMCs. Ensuing aggravation of slow conduction may contribute to the precipitation of strain-related arrhythmias in fibrotically remodelled hearts. [ABSTRACT FROM AUTHOR]

Aims The majority of patients diagnosed with arrhythmogenic right ventricular cardiomyopathy (ARVC) have mutations in genes encoding desmosomal proteins, raising the possibility that abnormal intercellular adhesion plays an important role in disease pathogenesis. We characterize cell mechanical properties and molecular responses to oscillatory shear stress in cardiac myocytes expressing mutant forms of the desmosomal proteins, plakoglobin and plakophilin, which are linked to ARVC in patients. Methods and results Cells expressing mutant plakoglobin or plakophilin showed no differences in cell–cell adhesion relative to controls, while knocking down these proteins weakened cell–cell adhesion. However, cells expressing mutant plakoglobin failed to increase the amount of immunoreactive signal for plakoglobin or N-cadherin at cell–cell junctions in response to shear stress, as seen in control cells. Cells expressing mutant plakophilin exhibited a similar attenuation in the shear-induced increase in junctional plakoglobin immunoreactive signal in response to shear stress, suggesting that the phenotype is independent of the type of mutant protein being expressed. Cells expressing mutant plakoglobin also showed greater myocyte apoptosis compared with controls. Apoptosis rates increased greatly in response to shear stress in cells expressing mutant plakoglobin, but not in controls. Abnormal responses to shear stress in cells expressing either mutant plakoglobin or plakophilin could be reversed by SB216763, a GSK3β inhibitor. Conclusions Desmosomal mutations linked to ARVC do not significantly affect cell mechanical properties, but cause myocytes to respond abnormally to mechanical stress through a mechanism involving GSK3β. These results may help explain why patients with ARVC experience disease exacerbations following strenuous exercise. [ABSTRACT FROM AUTHOR]

Aims Elevation of intracellular Na in the failing myocardium contributes to contractile dysfunction, the negative force–frequency relationship, and arrhythmias. Although phospholemman (PLM) is recognized to form the link between signalling pathways and Na/K pump activity, the possibility that defects in its regulation contribute to elevation of intracellular Na has not been investigated. Our aim was to test the hypothesis that the prevention of PLM phosphorylation in a PLM3SA knock-in mouse (in which PLM has been rendered unphosphorylatable) will exacerbate cardiac hypertrophy and cellular Na overload. Testing this hypothesis should determine whether changes in PLM phosphorylation are simply bystander effects or are causally involved in disease progression. Methods and results In wild-type (WT) mice, aortic constriction resulted in hypophosphorylation of PLM with no change in Na/K pump expression. This under-phosphorylation of PLM occurred at 3 days post-banding and was associated with a progressive decline in Na/K pump current and elevation of [Na]i. Echocardiography, morphometry, and pressure-volume (PV) catheterization confirmed remodelling, dilation, and contractile dysfunction, respectively. In PLM3SA mice, expression of Na/K ATPase was increased and PLM decreased such that net Na/K pump current under quiescent conditions was unchanged (cf. WT myocytes); [Na+]i was increased and forward-mode Na/Ca exchanger was reduced in paced PLM3SA myocytes. Cardiac hypertrophy and Na/K pump inhibition were significantly exacerbated in banded PLM3SA mice compared with banded WT. Conclusions Decreased phosphorylation of PLM reduces Na/K pump activity and exacerbates Na overload, contractile dysfunction, and adverse remodelling following aortic constriction in mice. This suggests a novel therapeutic target for the treatment of heart failure. [ABSTRACT FROM PUBLISHER]

Aims Atrial fibrillation (AF) is the most common cardiac arrhythmia and often involves reentrant electrical activation (e.g. spiral waves). Drug therapy for AF can have serious side effects including proarrhythmia, while electrical shock therapy is associated with discomfort and tissue damage. Hypothetically, forced expression and subsequent activation of light-gated cation channels in cardiomyocytes might deliver a depolarizing force sufficient for defibrillation, thereby circumventing the aforementioned drawbacks. We therefore investigated the feasibility of light-induced spiral wave termination through cardiac optogenetics. Methods and results Neonatal rat atrial cardiomyocyte monolayers were transduced with lentiviral vectors encoding light-activated Ca2+-translocating channelrhodopsin (CatCh; LV.CatCh∼eYFP↑) or eYFP (LV.eYFP↑) as control, and burst-paced to induce spiral waves rotating around functional cores. Effects of CatCh activation on reentry were investigated by optical and multi-electrode array (MEA) mapping. Western blot analyses and immunocytology confirmed transgene expression. Brief blue light pulses (10 ms/470 nm) triggered action potentials only in LV.CatCh∼eYFP↑-transduced cultures, confirming functional CatCh-mediated current. Prolonged light pulses (500 ms) resulted in reentry termination in 100% of LV.CatCh∼eYFP↑-transduced cultures (n = 31) vs. 0% of LV.eYFP↑-transduced cultures (n = 11). Here, CatCh activation caused uniform depolarization, thereby decreasing overall excitability (MEA peak-to-peak amplitude decreased 251.3 ± 217.1 vs. 9.2 ± 9.5 μV in controls). Consequently, functional coresize increased and phase singularities (PSs) drifted, leading to reentry termination by PS–PS or PS–boundary collisions. Conclusion This study shows that spiral waves in atrial cardiomyocyte monolayers can be terminated effectively by a light-induced depolarizing current, produced by the arrhythmogenic substrate itself, upon optogenetic engineering. These results provide proof-of-concept for shockless defibrillation. [ABSTRACT FROM PUBLISHER]

Aims Dysregulation of autonomic nervous system activity can trigger ventricular arrhythmias and sudden death in patients with heart failure. N-type Ca2+ channels (NCCs) play an important role in sympathetic nervous system activation by regulating the calcium entry that triggers release of neurotransmitters from peripheral sympathetic nerve terminals. We have investigated the ability of NCC blockade to prevent lethal arrhythmias associated with heart failure. Methods and results We compared the effects of cilnidipine, a dual N- and L-type Ca2+ channel blocker, with those of nitrendipine, a selective L-type Ca2+ channel blocker, in transgenic mice expressing a cardiac-specific, dominant-negative form of neuron-restrictive silencer factor (dnNRSF-Tg). In this mouse model of dilated cardiomyopathy leading to sudden arrhythmic death, cardiac structure and function did not significantly differ among the control, cilnidipine, and nitrendipine groups. However, cilnidipine dramatically reduced arrhythmias in dnNRSF-Tg mice, significantly improving their survival rate and correcting the imbalance between cardiac sympathetic and parasympathetic nervous system activity. A β-blocker, bisoprolol, showed similar effects in these mice. Genetic titration of NCCs, achieved by crossing dnNRSF-Tg mice with mice lacking CACNA1B, which encodes the α1 subunit of NCCs, improved the survival rate. With restoration of cardiac autonomic balance, dnNRSF-Tg;CACNA1B+/− mice showed fewer malignant arrhythmias than dnNRSF-Tg;CACNA1B+/+ mice. Conclusions Both pharmacological blockade of NCCs and their genetic titration improved cardiac autonomic balance and prevented lethal arrhythmias in a mouse model of dilated cardiomyopathy and sudden arrhythmic death. Our findings suggest that NCC blockade is a potentially useful approach to preventing sudden death in patients with heart failure. [ABSTRACT FROM PUBLISHER]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The Netherlands Cardio Vascular Research Initiative (CVON): the Dutch Heart Foundation, Dutch Federation of University Medical Center, the Netherlands Organization for Health Re-search and Development and the Royal Netherlands Academy of Sciences Introduction Sudden cardiac death (SCD) is one of the severe manifestations in carriers with a phospholamban (PLN p.Arg14del) cardiomyopathy. Prediction whether a patient with this pathogenic variant will be at risk for SCD is difficult. PLN has an important role in cardiac calcium homeostasis as regulator of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). It has been shown that the p.Arg14del pathogenic variant leads to Ca2+ overload in cardiomyocytes. Recently, it was found that dilated cardiomyopathy (DCM) patients who have a polymorphism in histidine-rich calcium binding protein (HRC Ser96Ala, rs3745297), displayed an increased risk for malignant arrhythmias and SCD. HRC resides within the SR, where it acts as a regulator of Ca2+ homeostasis. This Ser96Ala gene variant is widespread, as 60% of the general population bears at least one copy of this allele. Objective To explore the effect of the HRC Ser96Ala polymorphism on ventricular arrhythmias and disease expression in PLN p.Arg14del pathogenic variant carriers. Methods 337 p.Arg14del patients were included into the study; divided into wildtype (WT) (n=134, 24 index patients), heterozygous for Ser96Ala variant (n=142, 30 index patients) and homozygous for Ser96Ala variant (n=61, 11 index patients). The study was conducted according to the Declaration of Helsinki. Blood samples were genotyped on the Infinium® Global Screening Array-24 v3.0. Clinical data were subtracted from health records. Results In total 23% of PLN variant carriers were diagnosed with DCM while 11% of the variant carriers were diagnosed with arrhythmogenic cardiomyopathy. A significant difference in age of presentation (p=0.019) of p.Arg14del patients diagnosed with a DCM phenotype was found in homozygous HRC variant carriers (median 43 years, [36-47.3], n=8) compared to WT (median 49 years, [41-56.8], n=24) and heterozygous variant carriers (median 58.5 years, [51-66.5], n=22). No significant differences between the 3 groups were detected in manifestations of premature ventricular contractions (n=188, p=0.203), non-sustained ventricular tachycardia (n=248, p=0.314) and appropriate ICD shocks (n=308, p=0.901). Conclusion Although a significant difference in disease onset was found in PLN p.Arg14del patients with DCM who were homozygous for the HRC polymorphism, no correlations with arrhythmogenic parameters were found between patients with and without the HRC polymorphism. Therefore, we conclude that presence of the HRC polymorphism is not a discriminative predictor for arrhythmogenic events in PLN p.Arg14del. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Research Foundation - Flanders (FWO) Introduction SCN5A encodes the α-subunit of voltage-gated cardiac sodium channel Nav1.5. Mutations in SCN5A are identified in about 20% of patients with Brugada syndrome (BrS), an inherited cardiac arrhythmia. We have identified an SCN5A founder mutation (c.4813+3_4813+6dupGGGT), leading to a loss-of-function of Nav1.5 in 25 different families. Mutation carriers show variable expression of the phenotype: from asymptomatic to syncopes and sudden cardiac death. We used induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) to investigate the underlying pathophysiology. Material & Methods Dermal fibroblasts of six patients with different disease severity, and two unrelated healthy control individuals were reprogrammed using a commercially available reprogramming kit. iPSC-CMs were differentiated following a published protocol. We performed several differentiation rounds and investigated expression of cardiac markers using qPCR and immunocytochemistry and electrophysiological properties using patch-clamping. Results All iPSC-CMs expressed the tested markers. We observed reduction in sodium current density in patient iPSC-CMs compared to the control cells. However, our data display variability in AP characteristics between the differentiation batches, as well as between clones generated from one donor. Conclusions We established iPSC-CM models for a unique Belgian SCN5A founder mutation. Despite the observed variability, we could detect expected differences in electrophysiological properties of patient cells compared to controls. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Research, Development and Innovation Office Introduction Polycystic ovary syndrome (PCOS) is a multifactorial, endocrine disease associated with metabolic disturbances (e.g. hyperinsulinemia, insulin resistance) and increased cardiovascular risk. Recent data strongly suggest that different QT variability parameters characterizing cardiac repolarization instability represent novel markers in proarrhythmic risk assessment. Purpose In the present study we investigated ECG repolarization parameters, including QT variability parameters in patients with polycystic ovary syndrome. Methods Fifty-five PCOS patients (age: 29±6 years) and 55 age-matched healthy volunteers (age: 29±10 years) were enrolled in the study. Five-minute 12-lead resting electrocardiograms were recorded, the ECGs were digitized and evaluated off-line using the Cardiosys-A01 system (Cardiosys-A01, MDE Heidelberg GMBH, Heidelberg, Germany). The following parameters were determined: the frequency corrected QT interval (QTc) using Bazett's, Fridericia, Framingham and the Hodges formulas; QT dispersion (QTd) and T wave peak-to-end distance (Tpeak-Tend). Among QT variability parameters we analyzed the QT variance (QTv), the QT variability index (QTVI), the short-term beat-to-beat QT and RR interval variability (STV-QT, STV-RR) based on constructed Poincaré plots and the variability ratio (VR). Results The RR interval did not differ significantly in PCOS patients compared to controls (821±129 ms vs. 847±99 ms), however the QT interval (373±30 ms vs. 391±27 ms, p<0.01), the QTc calculated with Bazett's, Framingham, Fridericia and Hodges correction formulas (QTc Bazett's: 413±18 ms vs. 426±21 ms, p<0.01) and the Tpeak-Tend intervals were significantly shorter (76±10 ms vs. 83±12 ms, p<0.01). The QTd, QTv, and STV-RR did not differ significantly. However, the VR (0.3±0.4 vs. 0.2±0.2, p<0.05), the QTVI (-0,9±0.5 vs. -1,3±0.4, p<0.001), and importantly, the STV-QT were significantly higher in PCOS patients compared to controls (4.0±0.9 ms vs. 3.2±0.9 ms, p<0.0001). Conclusion Some of the alterations in repolarization parameters and the significant increase in the short-term beat-to-beat QT interval variability and the QT variability index may indicate increased repolarization instability in patients with polycystic ovary syndrome compared to age-matched controls, however, further studies are needed to establish the exact relation of this finding to increased arrhythmia propensity in this population. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft, German Centre for Cardiovascular Research (DZHK) Background Cardiac arrhythmias, such as atrial fibrillation (AF), have been linked to the remodeling of membrane receptors and alterations in cAMP-dependent regulation of calcium handling mechanisms. For instance, decreased L-type calcium channel (LTCC) current density but upregulated ryanodine receptor 2 (RyR2) are major hallmarks of AF. Furthermore, adenosine A2A receptor (A2AR) stimulation increases calcium waves without affecting LTCC and serotonin (5-HT) receptors activation exerts stronger control over myofilaments than over RyR2 function. However, up to date no study has elucidated how the increase on cAMP upon different G-protein-coupled receptors (GPCR) stimulation can lead to different physiological compartmentalized responses. The aim of this study was to investigate the effects of various GPCRs on cAMP levels in different cellular compartments in human atrial myocytes from control patients in sinus rhythm (Ctl), and how these compartmentalized effects are altered in AF. Furthermore, alterations in downstream cAMP level control by phosphodiesterases (PDEs) between patient groups were investigated to further elucidate functional differences. Methods Atrial myocytes were isolated from tissues of 66 AF and 80 Ctl patients. Cells were then transduced with adenoviruses (Epac1-camps, pm-Epac1-camps and Epac1-JNC) and cultured for 48 hours to express the Förster-resonance energy transfer (FRET)-based cAMP sensor in the cytosolic, membrane and RyR2 nanodomains, respectively. FRET was then used to measure cAMP in 532 isolated human atrial myocytes. Stimulation with β-adrenergic agonist Isoprenaline (ISO, 100nM) was used and compared with 5-HT (100µM) and A2AR (with CGS, 200nM) stimulation. Additionally, a nonspecific PDE inhibitor (IBMX, 100µM) was applied, as well as PDE3 (Cilostamide, 1µM) and PDE4 (Ro 20-1724, 10µM) specific inhibitors. Results A desensitization to β-adrenergic receptor stimulation in AF myocytes was exclusively found in the cytosol, while no difference was seen in neither the RyR2 nor LTCC compartment in AF versus Ctl. Similar effects were observed upon 5-HT stimulation with a significant desensitization in the cytosol, and no difference in the RyR2 compartment. On the contrary, AF myocytes displayed a significantly higher increase in cAMP levels compared to Ctl myocytes in the cytosol upon A2ARs stimulation. Importantly, no effect on cAMP levels was observed in the LTCC compartment after A2ARs or 5-HT stimulation. However, PDE3 inhibition on top of 5-HT stimulation showed a significantly smaller effect on cAMP levels in AF myocytes within the LTCC compartment. Conclusions Collectively, our data show that cAMP levels are highly compartmentalized in human atrial myocytes and differentially regulated by different GPCRs. Furthermore, PDEs are to a certain extent responsible for the compartmentalized effects of the different GPCRs. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: None. Light pollution disrupts circadian rhythms and increases the development of obesity, but their connection to cardiovascular disease, in particular to malignant arrhythmias, is not well known. Herein, we investigated if female rats exposed to continuous light and high-sucrose diet had altered myocardial gene transcripts and/or protein expression which affects arrhythmogenesis. We then assessed if Omacor® supplementation benefitted affected rats. In a first experiment, adult female spontaneously hypertensive (SHR) and normotensive Wistar rats (WR) were housed under standard 12 h/12 h light/dark cycles or exposed to 6-weeks continuous 300 lux light for 24 h. In a second experiment, adult female WR rats received sucrose diet (30% sucrose solution) continuously (24 h) performed for 8 weeks. Half the rats from both experiments were throughout the experiment treated with 200 mg/100g b.w. Omacor®. Continuous light resulted in higher rat vulnerability to malignant ventricular fibrillation (VF). This was linked with myocardial connexin-43 (Cx43) down-regulation and deteriorated intercellular electrical coupling, due in part to increased pro-inflammatory NF-κB and iNOS transcripts and decreased sarcoplasmic reticulum Ca 2+ ATPase transcripts. Omacor® treatment increased the electrical threshold to induce the VF linked with amelioration of myocardial Cx43 mRNA and Cx43 protein levels and the suppression of NF-κB and iNOS. 8 weeks lasting intake of 30% sucrose solution downregulated Cx43 and PKCε signaling along with an upregulation of myocardial PKCδ and miR-30a rendering the heart prone to ventricular arrhythmias. There was a clear benefit of Omacor® supplementation due to their antiarrhythmic effects associated with the attenuation of myocardial Cx43, PKC, and miR-30a abnormalities. This indicates that rat exposure to continuous light and high-sucrose result in deleterious cardiac alterations jeopardizing intercellular Cx43 channel-mediated electrical communication, thereby increasing the risk of malignant arrhythmias. The adverse effects were attenuated by treatment with Omacor®, thus supporting its potential benefit and the relevance of monitoring omega-3 index in human populations at risk. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): South-Eastern Norway Regional Health Authority Introduction Ventricular arrhythmias are common during the early phase of reperfusion after an ischemic event. Spontaneous diastolic release of Ca2+ from the sarcoplasmic reticulum (SR) contribute to arrhythmias in this stage, and reactive oxygen species (ROS) have been implicated in the process, through destabilization of key Ca2+ handling proteins. Specifically, ROS has been shown to activate the Ca2+/calmodulin-dependent protein kinase II (CaMKII), by oxidation of two methionines at positions 281/282 in the regulatory domain. This activation pathway contributes to Ca2+-dependent arrhythmia mechanisms in atrial fibrillation (AF), heart failure (HF) and diabetes. However, the role of oxidized CaMKII (Ox-CaMKII) in ventricular arrhythmias during early reperfusion has not been directly tested. We aimed to investigate the importance of ox-CaMKII for diastolic Ca2+ release events and arrhythmias in the early phase of reperfusion. Methods We evaluated the propensity for early reperfusion arrhythmias in Langendorff-perfused hearts from mice carrying CaMKII resistant to oxidation (homozygous CaMKIIδ M281/282V mice) and wild type control mice (WT). The effect of beta-adrenoceptor stimulation, indirect inhibition of CaMKII via calmodulin antagonism, and antioxidant therapy was evaluated by the addition of isoprenaline, KN93 and N-acetylcysteine (NAC), respectively. Diastolic Ca2+ release events were recorded by confocal line scan imaging of isolated cardiomyocytes transiently exposed to hypoxia and ischemia-like conditions in the presence or absence of isoprenaline, and effects on Ca2+ handling proteins were assessed by immunoblotting. Results Langendorff-perfused hearts from CaMKIIδ M281/282V mice showed no significant difference in the frequency of ventricular arrhythmias compared to WT during the early reperfusion phase, neither in absence, nor presence of beta-adrenoceptor stimulation with isoprenaline. The incidence of ventricular arrhythmias increased significantly upon beta-adrenoceptor stimulation but was prevented by the antioxidative agent NAC and reduced with CaMKII-inhibition with KN93 when tested in WT hearts. Immunoblotting of left ventricular tissue from perfused hearts revealed no significant differences in the abundance or phosphorylation levels of Ca2+-handling proteins. Ca2+ imaging experiments showed no differences between cardiomyocytes from CaMKIIδ M281/282V and WT mice with regard to incidence of spontaneous transients, Ca2+ waves or Ca2+ sparks, neither in absence, nor in presence of isoprenaline. Conclusions This study demonstrates the importance of both ROS and CaMKII activation in the development of ventricular arrhythmias in the early phase of reperfusion. However, genetic ablation of CaMKII oxidation did not protect against early reperfusion arrhythmias, indicating that Ox-CaMKII is a not a critical factor in this process in Langendorff-perfused hearts. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Instituto de Salud Carlos III CIBERCV Introduction Cardiac arrhythmias can originate as a consequence of the proarrhythmic effects of some therapeutical regenerative products. In vitro studies have demonstrated different levels of electrophysiological complexity but these models usually lack of direct translation into the clinical practice. Objective To develop an arrhythmia complexity evaluation method that enables to characterize electrophysiological complexity in translational scenarios. Methodology: To build a new evaluation method, frequency and rotor biomarkers from in vitro cell cultures presenting fibrillatory activity (N=15) were evaluated using optical mapping recordings (Rhod-2AM Ca2+ transient dye, 30-minute incubation, excited with green light). The patterns observed at in vitro level were analyzed and characterized by means of frequency and rotor biomarkers. Next, we performed a forward translational study to evaluate these electrophysiological biomarkers at a clinical level. In 206 patients with atrial fibrillation (AF) with non-invasive body surface recordings using the Acorys system from Corify (67 electrodes, 4 second segments) (STRATIFY-AF study) were evaluated to analyze the same frequency and rotor biomarkers previously explored in vitro. Moreover, we build a complexity score that described from 0 to 1 the complexity at electrophysiological level, where 0 corresponded to simple scenarios and 1 to complex arrhythmic patterns. Patients were followed for 12 months to evaluate long term evolution. Results Complexity evaluation method identified the highest dominant frequency, median frequency and rotor time as the most important predictors for arrhythmia complexity definition. In vitro cultures results showed that samples presenting higher dominant frequency were also associated with small dominant frequency area extension and lower rotor time maintenance, i.e. with a high value of the complexity biomarker (Figure 1B). At clinical level, patients with high values of the complexity score were identified with Persistent AF patients showing complex patterns in whom AF recurred after ablation at 12 months (Figure 1D). Conclusions We developed a translational approach that enables to characterize arrhythmia complexity in different frameworks, which is crucial to evaluate potential undesirable effects of therapeutical regenerative products during the complete development and validation of the product. Open in new tab Download slide In vitro and patient complexity examples [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): RICERCA CORRENTE, Italian Ministry of Health Background Brugada syndrome (BrS) is a cardiac arrhythmia associated with an increased risk of sudden cardiac death (SCD). BrS is considered a genetic disorder, and the most commonly mutated gene is SCN5A, which encodes the alpha subunit of the voltage-gated cardiac sodium channel (NaV1.5). Mutations of SCN5A generally cause impairment of NaV1.5 function, resulting in alteration of the action potential. However, mutations of SCN5A are responsible for only 30% of BrS cases. Therefore, it is conceivable that other mechanisms such as post-translational modifications (PTMs) could affect NaV1.5 activity. Among others, sialylation may alter ion channel activity by carrying a sugar with a negative charge. Alterations in sialylation have previously been described in several cardiovascular diseases, including myocardial infarction, Chagas disease, and congenital disorders of glycosylation, a family of diseases affecting the heart. For these reasons, the aim was to study alterations in sialylation in BrS patients to get new information about the pathogenesis of BrS. Materials and Methods Peripheral blood mononuclear cells (PBMCs) were collected from BrS patients and healthy controls. To characterize the protein sialylation status of PBMCs, SNA lectin, which is sialic acid-binding proteins, was used by Western blot and flow cytometry. Gene expression of enzymes involved in the biosynthesis, activation, transfer, degradation, and recycling of sialic acid was examined in PBMCs by real-time PCR. Results The extracellular and intracellular sialylation levels of PBMCs differed between BrS patients and controls. In particular, CD3+/CD4+ T cells exhibited a lower significant amount of sialic acid. Moreover, gene expression of enzymes involved in the biosynthesis and activation of sialic acid was downregulated in patients compared to controls. In contrast, the sialyltransferases St3Gal1, St3Gal4, and St6Gal2, enzymes responsible for binding sialic acid to the protein surface, showed significant enzyme-specific differences between BrS patients and controls. Moreover, the levels of sialyltransferases and sialylated proteins are inversely correlated with the epicardial area of the cardiac pathological substrate and with potential duration, two clinical indicators of BrS severity. Discussion These preliminary results demonstrate the involvement of sialylation in BrS syndrome and suggest that impairment of the sialylation process may be associated with the onset and/or manifestation of BrS. In addition, biochemical alteration of cells of the BrS immune system was reported for the first time, supporting the hypothesis that BrS is a systemic disease whose final manifestation is an increased risk of SCD. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Marie Skłodowska-Curie Actions - Individual Fellowship Background The cardiac sodium channel encoded by SCN5A gene undergoes fetal-to-adult isoform switch postnatally, regulated by alternative splicing. Various mutations in SCN5A associated with severe cardiac arrhythmia have been previously studied using cardiomyocytes derived from human induced pluripotent stem-cells (hiPSC-CMs), demonstrating the importance of a human cardiac cellular context for such analysis. Mutations in the adult isoform of SCN5A are however of difficult evaluation in hiPSC-CMs, because of their typical immature/fetal-like characteristics. Combining hiPSC-CMs in 3-dimensional microtissues together with hiPSC-derived cardiac fibroblasts and endothelial cells induced maturation in gene expression and functional properties, including electrophysiology. Purpose We investigated whether the maturation promoted by the microtissue system could reveal the functional phenotype of a mutation in the adult isoform of SCN5A. Methods We derived hiPSC-CMs from a cardiac arrhythmia patient carrying two compound mutations in SCN5A: p.W156X in exon 4 and p.R225W in the adult splicing variant of exon 6 (exon 6B). Using CRISPR/Cas9, we corrected exon 4 mutation to investigate specific effects of exon 6B mutation on sodium current by single-cell patch clamp. HiPSC-CMs were matured in tricell-type cardiac microtissues and analysed after dissociation. The mechanism underlying exon 6B expression was investigated by overexpressing or knocking-out the alternative splicing regulator MBNL1 in hiPSC-CMs. Results ddPCR analysis showed low expression of exon 6B-containing transcripts in hiPSC-CMs, accompanied by no effect of exon 6B mutation on the sodium current. Instead, maturing hiPSC-CMs in cardiac microtissues promoted SCN5A exon 6B expression and revealed the contribution of both SCN5A mutations to the diseased cellular electrophysiology. MBNL1 was upregulated in microtissues and indeed its overexpression was sufficient to increase exon 6B levels in hiPSC-CMs, while lack of MBNL1 prevented the switch of SCN5A splicing isoforms. Conclusions Our data demonstrate that a maturation of hiPSC-CMs is required to express the adult SCN5A isoform. This was achieved in the microtissue system via upregulation of MBNL1, and allowed to reveal SCN5A mutation contributions, by dissecting changes in ionic current that cause adult arrhythmic disease phenotypes in humans. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Junta de Andalucia Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia in humans. Genetic and genomic analyses have recently demonstrated that the homeobox transcription factor Pitx2 plays a fundamental role regulating expression of distinct growth factors, microRNAs and ion channels leading to morphological and molecular alterations that promote the onset of AF. Here we address the plausible contribution of long non-coding (lnc)RNAs within the Pitx2>Wnt>miRNA signaling pathway. In silico analyses of annotated lncRNAs in the vicinity of the Pitx2, Wnt8 and Wnt11 chromosomal loci identified five novel lncRNAs with differential expression during cardiac development. Importantly, three of them, Walaa, Walras and Wallrd, are evolutionarily conserved in humans and displayed preferential atrial expression during embryogenesis. In addition, Walrad displayed moderate expression during embryogenesis but was more abundant in the right atrium. Walaa, Walras and Wallrd were distinctly regulated by Pitx2, Wnt8, and Wnt11, and Wallrd was severely elevated in conditional atrium-specific Pitx2-deficient mice. Furthermore, pro-arrhythmogenic and pro-hypertrophic substrate administration to primary cardiomyocyte cell cultures consistently modulate expression of these lncRNAs, supporting distinct modulatory roles of the AF cardiovascular risk factors in the regulation of these lncRNAs. Walras affinity pulldown assays revealed its association with distinct cytoplasmic and nuclear proteins previously involved in cardiac pathophysiology, while loss-of-function assays further support a pivotal role of this lncRNA in cytoskeletal organization. We propose that lncRNAs Walaa, Walras and Wallrd, distinctly regulated by Pitx2>Wnt>miRNA signaling and pro-arrhythmogenic and pro-hypertrophic factors, are implicated in atrial arrhythmogenesis, and Walras additionally in cardiomyocyte cytoarchitecture. [ABSTRACT FROM AUTHOR]

Aims Fibroblasts, which play an important role in cardiac function/dysfunction, including arrhythmogenesis, have voltage-dependent (Kv) currents of unknown importance. Here, we assessed the differential expression of Kv currents between atrial and ventricular fibroblasts from control dogs and dogs with an atrial arrhythmogenic substrate caused by congestive heart failure (CHF). Methods and results Left atrial (LA) and ventricular (LV) fibroblasts were freshly isolated from control and CHF dogs (2-week ventricular tachypacing, 240 bpm). Kv currents were measured with whole-cell voltage-clamp, mRNA by quantitative polymerase chain reaction (qPCR) and fibroblast proliferation by 3H-thymidine incorporation. Robust voltage-dependent tetraethylammonium (TEA)-sensitive K+ currents (IC50 ∼1 mM) were recorded. The morphologies and TEA responses of LA and LV fibroblast Kv currents were similar. LV fibroblast Kv-current densities were significantly greater than LA, and Kv-current densities were significantly less in CHF than control. The mRNA expression of Kv-channel subunits Kv1.5 and Kv4.3 was less in LA vs. LV fibroblasts and was down-regulated in CHF, consistent with K+-current recordings. Ca2+-dependent K+-channel subunit (KCa1.1) mRNA and currents were less expressed in LV vs. LA fibroblasts. Inhibiting LA fibroblast K+ current with 1 mmol/L of TEA or KCa1.1 current with paxilline increased proliferation. Conclusions Fibroblast Kv-current expression is smaller in CHF vs. control, as well as LA vs. LV. KCa1.1 current is greater in LA vs. LV. Suppressing Kv current with TEA enhances fibroblast proliferation, suggesting that Kv current might act to check fibroblast proliferation and that reduced Kv current in CHF may contribute to fibrosis. Fibroblast Kv-current remodelling may play a role in the atrial fibrillation (AF) substrate; modulating fibroblast K+ channels may present a novel strategy to prevent fibrosis and AF. [ABSTRACT FROM PUBLISHER]

Despite major advances in the treatment of cardiac diseases, there is still a great need for drugs capable of counteracting the deterioration of cardiac muscle function in congestive heart failure. The role of misfolded protein accumulation as a causal event in the physiopathology of common cardiac diseases is an important emerging concept. Indeed, diverse stress conditions, including mechanical stretching and oxidative stress, can induce misfolded protein accumulation, causing cardiomyocyte death. Cells react to these stress conditions by activating molecular chaperones, a class of proteins that represents an endogenous salvage machinery, essential for rescuing physiological cell functions and sustaining cell survival. Chaperones, also known as heat shock proteins (Hsps), prevent accumulation of damaged proteins by promoting either their refolding or degradation via the proteasome or the autophagosome systems. In addition, molecular chaperones play a key role in intracellular signalling by controlling conformational changes required for activation/deactivation of signalling proteins, and their assembly in specific signalosome complexes. The key role of molecular chaperones in heart function is highlighted by the fact that a number of genetic mutations in chaperone proteins result in different forms of cardiomyopathies. Moreover, a considerable amount of experimental evidence indicates that increasing expression of chaperone proteins leads to an important cardio-protective role in ischaemia/reperfusion injury, heart failure, and arrhythmia, implicating these molecules as potential innovative therapeutic agents. [ABSTRACT FROM PUBLISHER]

Aims Cardiac function depends on the highly regulated and co-ordinate activity of a large ensemble of potassium channels that control myocyte repolarization. While voltage-gated K+ channels have been well characterized in the heart, much less is known about regulation and/or targeting of two-pore K+ channel (K2P) family members, despite their potential importance in modulation of heart function. Methods and results Here, we report a novel molecular pathway for membrane targeting of TREK-1, a mechano-sensitive K2P channel regulated by environmental and physical factors including membrane stretch, pH, and polyunsaturated fatty acids (e.g. arachidonic acid). We demonstrate that βIV-spectrin, an actin-associated protein, is co-localized with TREK-1 at the myocyte intercalated disc, associates with TREK-1 in the heart, and is required for TREK-1 membrane targeting. Mice expressing βIV-spectrin lacking TREK-1 binding (qv4J) display aberrant TREK-1 membrane localization, decreased TREK-1 activity, delayed action potential repolarization, and arrhythmia without apparent defects in localization/function of other cardiac potassium channel subunits. Finally, we report abnormal βIV-spectrin levels in human heart failure. Conclusions These data provide new insight into membrane targeting of TREK-1 in the heart and establish a broader role for βIV-spectrin in organizing functional membrane domains critical for normal heart function. [ABSTRACT FROM PUBLISHER]

Background A recent genome-wide association study identified a susceptibility locus for atrial fibrillation at the KCNN3 gene. Since the KCNN3 gene encodes for a small conductance calcium-activated potassium channel, we hypothesized that overexpression of the SK3 channel increases susceptibility to cardiac arrhythmias. Methods and results We characterized the cardiac electrophysiological phenotype of a mouse line with overexpression of the SK3 channel. We generated homozygote (SK3T/T) and heterozygote (SK3+/T) mice with overexpression of the channel and compared them with wild-type (WT) controls. We observed a high incidence of sudden death among SK3T/T mice (7 of 19 SK3T/T mice). Ambulatory monitoring demonstrated that sudden death was due to heart block and bradyarrhythmias. SK3T/T mice displayed normal body weight, temperature, and cardiac function on echocardiography; however, histological analysis demonstrated that these mice have abnormal atrioventricular node morphology. Optical mapping demonstrated that SK3T/T mice have slower ventricular conduction compared with WT controls (SK3T/T vs. WT; 0.45 ± 0.04 vs. 0.60 ± 0.09 mm/ms, P = 0.001). Programmed stimulation in 1-month-old SK3T/T mice demonstrated inducible atrial arrhythmias (50% of SK3T/T vs. 0% of WT mice) and also a shorter atrioventricular nodal refractory period (SK3T/T vs. WT; 43 ± 6 vs. 52 ± 9 ms, P = 0.02). Three-month-old SK3T/T mice on the other hand displayed a trend towards a more prolonged atrioventricular nodal refractory period (SK3T/T vs. WT; 61 ± 1 vs. 52 ± 6 ms, P = 0.06). Conclusion Overexpression of the SK3 channel causes an increased risk of sudden death associated with bradyarrhythmias and heart block, possibly due to atrioventricular nodal dysfunction. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is a prevalent cardiac arrhythmia with a significant genetic component. In recent years, familial and population-based genetic studies in AF have led to the emergence of transcription factors as potentially important contributors to arrhythmia susceptibility. Further evidence to implicate transcription factors in AF has come from studies in animal models, which demonstrate that these proteins play critical roles during AF-related atrial remodelling. Transcription factors have the potential to create a pro-arrhythmogenic substrate in the pulmonary veins and the atrium. However, further research is necessary to fully characterize the mechanistic links between these proteins and AF pathogenesis. In the future, such studies could potentially lead to the development of novel therapies for the arrhythmia. This review focuses on the association between transcription factors and AF. [ABSTRACT FROM AUTHOR]

Aims Alterations in cardiac acid–base balance can produce profound impact on excitation–contraction coupling and precipitate cardiac dysfunction and arrhythmias. A member of the solute carrier (SLC) family, Slc26a6, has been shown to be a chloride-hydroxyl exchanger and the predominant chloride-bicarbonate exchanger in the mouse heart. However, the exact isoforms and functional characteristics of cardiac Slc26a6 remain unknown. The objective of the present study is to determine the molecular identity of cardiac Slc26a6 isoforms, to examine their cellular expressions in the heart, and to test the function of Slc26a6 in cardiomyocytes. Methods and results We examined the expression and function of slc26a6 in mouse cardiomyocytes using RT–PCR, immunofluorescence confocal microscopy, and patch-clamp technique coupled with the fast solution exchange system. We identified four cardiac Slc26a6 isoforms, denoted C-a, C-b, C-c, and C-d, and detected significant expression of Slc26a6 in the plasma membrane of both atrial and ventricular myocytes. Isoforms C-a and C-b share the same sequence with the previously reported murine Slc26a6a and Slc26a6b, respectively. Isoform C-c lacks an alternate in-frame exon 12, whereas C-d is a C-terminal truncated form resulting from 102 bp exon insertion between exons 15 and 16 compared with C-b. Patch-clamp recordings demonstrated electrogenic Cl−/oxalate and electrogenic Cl−/HCO3− exchange activities in cardiomyocytes. Conclusion We demonstrate that cardiac myocytes express different isoforms of Slc26a6, which encode electrogenic Cl−/HCO3− and Cl−/oxalate exchangers. The electrogenic nature of the Cl−/HCO3− exchange of cardiac Slc26a6 suggests important roles in regulating acid–base balance in the heart. [ABSTRACT FROM PUBLISHER]

This review presents the roles of cardiac sodium channel NaV1.5 late current (late INa) in generation of arrhythmic activity. The assumption of the authors is that proper Na+ channel function is necessary to the maintenance of the transmembrane electrochemical gradient of Na+ and regulation of cardiac electrical activity. Myocyte Na+ channels’ openings during the brief action potential upstroke contribute to peak INa and initiate excitation–contraction coupling. Openings of Na+ channels outside the upstroke contribute to late INa, a depolarizing current that persists throughout the action potential plateau. The small, physiological late INa does not appear to be critical for normal electrical or contractile function in the heart. Late INa does, however, reduce the net repolarizing current, prolongs action potential duration, and increases cellular Na+ loading. An increase of late INa, due to acquired conditions (e.g. heart failure) or inherited Na+ channelopathies, facilitates the formation of early and delayed afterpolarizations and triggered arrhythmias, spontaneous diastolic depolarization, and cellular Ca2+ loading. These in turn increase the spatial and temporal dispersion of repolarization time and may lead to reentrant arrhythmias. [ABSTRACT FROM AUTHOR]

Aims In excitable cells, KIR2.x ion-channel-carried inward rectifier current (IK1) is thought to set the negative and stable resting membrane potential, and contributes to action potential repolarization. Loss- or gain-of-function mutations correlate with cardiac arrhythmias and pathological remodelling affects normal KIR2.x protein levels. No specific IK1 inhibitor is currently available for in vivo use, which severely hampers studies on the precise role of IK1 in normal cardiac physiology and pathophysiology. The diamine antiprotozoal drug pentamidine (P) acutely inhibits IK1 by plugging the cytoplasmic pore region of the channel. We aim to develop more efficient and specific IK1 inhibitors based on the P structure. Methods and results We analysed seven pentamidine analogues (PA-1 to PA-7) for IK1 blocking potency at 200 nM using inside-out patches from KIR2.1 expressing HEK-293 cells. PA-6 showed the highest potency and was tested further. PA-6 blocked KIR2.x currents of human and mouse with low IC50 values (12–15 nM). Modelling indicated that PA-6 had less electrostatic but more lipophilic interactions with the cytoplasmic channel pore than P, resulting in a higher channel affinity for PA-6 (ΔG −44.1 kJ/Mol) than for P (ΔG −31.7 kJ/Mol). The involvement of acidic amino acid residues E224 and E299 in drug-channel interaction was confirmed experimentally. PA-6 did not affect INav1.5, ICa-L, IKv4.3, IKv11.1, and IKv7.1/minK currents at 200 nM. PA-6 inhibited the inward (50 nM 40%; 100 nM 59%; 200 nM 77%) and outward (50 nM 40%; 100 nM 76%; 200 nM 100%) components of IK1 in isolated canine adult-ventricular cardiomyocytes (CMs). PA-6 prolonged action potential duration of CMs by 8 (n = 9), 26 (n = 5), and 34% (n = 11) at 50, 100, and 200 nM, respectively. Unlike P, PA-6 had no effect on KIR2.1 channel expression at concentrations from 0.1 to 3 μM. However, PA-6 at 10 μM increased KIR2.1 expression levels. Also, PA-6 did not affect the maturation of hERG, except when applied at 10 μM. Conclusion PA-6 has higher efficiency and specificity to KIR2.x-mediated current than P, lengthens action potential duration, and does not affect channel trafficking at concentrations relevant for complete IK1 block. [ABSTRACT FROM PUBLISHER]

Early afterdepolarizations (EADs) are secondary voltage depolarizations during the repolarizing phase of the action potential, which can cause lethal cardiac arrhythmias. The occurrence of EADs requires a reduction in outward current and/or an increase in inward current, a condition called reduced repolarization reserve. However, this generalized condition is not sufficient for EAD genesis and does not explain the voltage oscillations manifesting as EADs. Here, we summarize recent progress that uses dynamical theory to build on and advance our understanding of EADs beyond the concept of repolarization reserve, towards the goal of developing a holistic and integrative view of EADs and their role in arrhythmogenesis. We first introduce concepts from nonlinear dynamics that are relevant to EADs, namely, Hopf bifurcation leading to oscillations and basin of attraction of an equilibrium or oscillatory state. We then present a theory of phase-2 EADs in nonlinear dynamics, which includes the formation of quasi-equilibrium states at the plateau voltage, their stabilities, and the bifurcations leading to and terminating the oscillations. This theory shows that the L-type calcium channel plays a unique role in causing the nonlinear dynamical behaviours necessary for EADs. We also summarize different mechanisms of phase-3 EADs. Based on the dynamical theory, we discuss the roles of each of the major ionic currents in the genesis of EADs, and potential therapeutic targets. [ABSTRACT FROM PUBLISHER]

Aims Atrial fibrillation (AF) is the most common cardiac arrhythmia, and early-onset lone AF has been linked to mutations in genes encoding ion channels. Mutations in the pore forming subunit KV4.3 leading to an increase in the transient outward potassium current (Ito) have previously been associated with the Brugada Syndrome. Here we aim to determine if mutations in KV4.3 or in the auxiliary subunit K+ Channel-Interacting Protein (KChIP) 2 are associated with early-onset lone AF. Methods and results Two hundred and nine unrelated early-onset lone AF patients (<40 years) were recruited. The entire coding sequence of KCND3 and KCNIP2 was bidirectionally sequenced. One novel non-synonymous mutation A545P was found in KCND3 and was neither present in the control group (n = 432 alleles) nor in any publicly available database. The proband had onset of persistent AF at the age of 22, and no mutations in genes previously associated with AF were found. Electrophysiological analysis of KV4.3-A545P expressed in CHO-K1 cells, revealed that peak-current density was increased and the onset of inactivation was slower compared with WT, resulting in a significant gain-of-function both in the absence and the presence of KChIP2. Conclusion Gain-of-function mutations in KV4.3 have previously been described in Brugada Syndrome, however, this is the first report of a KV4.3 gain-of-function mutation in early-onset lone AF. This association of KV4.3 gain-of-function and early-onset lone AF further supports the hypothesis that increased potassium current enhances AF susceptibility. [ABSTRACT FROM AUTHOR]