Extracellular Kir2.1C122Y Mutant Upsets Kir2.1-PIP2 Bonds and Is Arrhythmogenic in Andersen-Tawil Syndrome.

BACKGROUND: Andersen-Tawil syndrome type 1 is a rare heritable disease caused by mutations in the gene coding the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys (cysteine)122-to-Cys154 disulfide bond in the channel structure is crucial for proper folding but has not been associated with correct channel function at the membrane. We evaluated whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing its open state. METHODS: We identified a Kir2.1 loss-of-function mutation (c.366 A>T; p.Cys122Tyr) in an ATS1 family. To investigate its pathophysiological implications, we generated an AAV9-mediated cardiac-specific mouse model expressing the Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments. RESULTS: Kir2.1C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking. Molecular dynamics predicted that the C122Y mutation provoked a conformational change over the 2000-ns simulation, characterized by a greater loss of hydrogen bonds between Kir2.1 and phosphatidylinositol 4,5-bisphosphate than wild type (WT). Therefore, the phosphatidylinositol 4,5-bisphosphate-binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch clamping, the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing phosphatidylinositol 4,5-bisphosphate concentrations. In addition, the Kir2.1C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and NaV1.5 proteins. CONCLUSIONS: The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential for the channel function. We demonstrate that breaking disulfide bonds in the extracellular domain disrupts phosphatidylinositol 4,5-bisphosphate-dependent regulation, leading to channel dysfunction and defects in Kir2.1 energetic stability. The mutation also alters functional expression of the NaV1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.

Practical approach for atrial cardiomyopathy characterization in patients with atrial fibrillation.

Atrial fibrillation (AF) causes progressive structural and electrical changes in the atria that can be summarized within the general concept of atrial remodeling. In parallel, other clinical characteristics and comorbidities may also affect atrial tissue properties and make the atria susceptible to AF initiation and its long-term persistence. Overall, pathological atrial changes lead to atrial cardiomyopathy with important implications for rhythm control. Although there is general agreement on the role of the atrial substrate for successful rhythm control in AF, the current classification oversimplifies clinical management. The classification uses temporal criteria and does not establish a well-defined strategy to characterize the individual-specific degree of atrial cardiomyopathy. Better characterization of atrial cardiomyopathy may improve the decision-making process on the most appropriate therapeutic option. We review current scientific evidence and propose a practical characterization of the atrial substrate based on 3 evaluation steps starting with a clinical evaluation (step 1), then assess outpatient complementary data (step 2), and finally include information from advanced diagnostic tools (step 3). The information from each of the steps or a combination thereof can be used to classify AF patients in 4 stages of atrial cardiomyopathy, which we also use to estimate the success on effective rhythm control.

The Kir2.1E299V mutation increases atrial fibrillation vulnerability while protecting the ventricles against arrhythmias in a mouse model of short QT syndrome type 3.

AIMS: Short QT syndrome type 3 (SQTS3) is a rare arrhythmogenic disease caused by gain-of-function mutations in KCNJ2, the gene coding the inward rectifier potassium channel Kir2.1. We used a multidisciplinary approach and investigated arrhythmogenic mechanisms in an in-vivo model of de-novo mutation Kir2.1E299V identified in a patient presenting an extremely abbreviated QT interval and paroxysmal atrial fibrillation. METHODS AND RESULTS: We used intravenous adeno-associated virus-mediated gene transfer to generate mouse models, and confirmed cardiac-specific expression of Kir2.1WT or Kir2.1E299V. On ECG, the Kir2.1E299V mouse recapitulated the QT interval shortening and the atrial-specific arrhythmia of the patient. The PR interval was also significantly shorter in Kir2.1E299V mice. Patch-clamping showed extremely abbreviated action potentials in both atrial and ventricular Kir2.1E299V cardiomyocytes due to a lack of inward-going rectification and increased IK1 at voltages positive to -80 mV. Relative to Kir2.1WT, atrial Kir2.1E299V cardiomyocytes had a significantly reduced slope conductance at voltages negative to -80 mV. After confirming a higher proportion of heterotetrameric Kir2.x channels containing Kir2.2 subunits in the atria, in-silico 3D simulations predicted an atrial-specific impairment of polyamine block and reduced pore diameter in the Kir2.1E299V-Kir2.2WT channel. In ventricular cardiomyocytes, the mutation increased excitability by shifting INa activation and inactivation in the hyperpolarizing direction, which protected the ventricle against arrhythmia. Moreover, Purkinje myocytes from Kir2.1E299V mice manifested substantially higher INa density than Kir2.1WT, explaining the abbreviation in the PR interval. CONCLUSION: The first in-vivo mouse model of cardiac-specific SQTS3 recapitulates the electrophysiological phenotype of a patient with the Kir2.1E299V mutation. Kir2.1E299V eliminates rectification in both cardiac chambers but protects against ventricular arrhythmias by increasing excitability in both Purkinje-fiber network and ventricles. Consequently, the predominant arrhythmias are supraventricular likely due to the lack of inward rectification and atrial-specific reduced pore diameter of the Kir2.1E299V-Kir2.2WT heterotetramer.

Kir2.1-NaV1.5 channelosome and its role in arrhythmias in inheritable cardiac diseases.

Sudden cardiac death in children and young adults is a relatively rare but tragic event whose pathophysiology is unknown at the molecular level. Evidence indicates that the main cardiac sodium channel (NaV1.5) and the strong inward rectifier potassium channel (Kir2.1) physically interact and form macromolecular complexes (channelosomes) with common partners, including adapter, scaffolding, and regulatory proteins that help them traffic together to their eventual membrane microdomains. Most important, dysfunction of either or both ion channels has direct links to hereditary human diseases. For example, certain mutations in the KCNJ2 gene encoding the Kir2.1 protein result in Andersen-Tawil syndrome type 1 and alter both inward rectifier potassium and sodium inward currents. Similarly, trafficking-deficient mutations in the gene encoding the NaV1.5 protein (SCN5A) result in Brugada syndrome and may also disturb both inward rectifier potassium and sodium inward currents. Moreover, gain-of-function mutations in KCNJ2 result in short QT syndrome type 3, which is extremely rare but highly arrhythmogenic, and can modify Kir2.1-NaV1.5 interactions in a mutation-specific way, further highlighting the relevance of channelosomes in ion channel diseases. By expressing mutant proteins that interrupt or modify Kir2.1 or NaV1.5 function in animal models and patient-specific pluripotent stem cell-derived cardiomyocytes, investigators are defining for the first time the mechanistic framework of how mutation-induced dysregulation of the Kir2.1-NaV1.5 channelosome affects cardiac excitability, resulting in arrhythmias and sudden death in different cardiac diseases.

Intermediate Molecular Phenotypes to Identify Genetic Markers of Anthracycline-Induced Cardiotoxicity Risk.

Cardiotoxicity due to anthracyclines (CDA) affects cancer patients, but we cannot predict who may suffer from this complication. CDA is a complex trait with a polygenic component that is mainly unidentified. We propose that levels of intermediate molecular phenotypes (IMPs) in the myocardium associated with histopathological damage could explain CDA susceptibility, so variants of genes encoding these IMPs could identify patients susceptible to this complication. Thus, a genetically heterogeneous cohort of mice (n = 165) generated by backcrossing were treated with doxorubicin and docetaxel. We quantified heart fibrosis using an Ariol slide scanner and intramyocardial levels of IMPs using multiplex bead arrays and QPCR. We identified quantitative trait loci linked to IMPs (ipQTLs) and cdaQTLs via linkage analysis. In three cancer patient cohorts, CDA was quantified using echocardiography or Cardiac Magnetic Resonance. CDA behaves as a complex trait in the mouse cohort. IMP levels in the myocardium were associated with CDA. ipQTLs integrated into genetic models with cdaQTLs account for more CDA phenotypic variation than that explained by cda-QTLs alone. Allelic forms of genes encoding IMPs associated with CDA in mice, including AKT1, MAPK14, MAPK8, STAT3, CAS3, and TP53, are genetic determinants of CDA in patients. Two genetic risk scores for pediatric patients (n = 71) and women with breast cancer (n = 420) were generated using machine-learning Least Absolute Shrinkage and Selection Operator (LASSO) regression. Thus, IMPs associated with heart damage identify genetic markers of CDA risk, thereby allowing more personalized patient management.

Extracellular cysteine disulfide bond break at Cys122 disrupts PIP2-dependent Kir2.1 channel function and leads to arrhythmias in Andersen-Tawil Syndrome.

Background: Andersen-Tawil Syndrome Type 1 (ATS1) is a rare heritable disease caused by mutations in the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys122-to-Cys154 disulfide bond in the Kir2.1 channel structure is crucial for proper folding, but has not been associated with correct channel function at the membrane. We tested whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing the open state of the channel. Methods and Results: We identified a Kir2.1 loss-of-function mutation in Cys122 (c.366 A>T; p.Cys122Tyr) in a family with ATS1. To study the consequences of this mutation on Kir2.1 function we generated a cardiac specific mouse model expressing the Kir2.1C122Y mutation. Kir2.1C122Y animals recapitulated the abnormal ECG features of ATS1, like QT prolongation, conduction defects, and increased arrhythmia susceptibility. Kir2.1C122Y mouse cardiomyocytes showed significantly reduced inward rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking ability and localization at the sarcolemma and the sarcoplasmic reticulum. Kir2.1C122Y formed heterotetramers with wildtype (WT) subunits. However, molecular dynamic modeling predicted that the Cys122-to-Cys154 disulfide-bond break induced by the C122Y mutation provoked a conformational change over the 2000 ns simulation, characterized by larger loss of the hydrogen bonds between Kir2.1 and phosphatidylinositol-4,5-bisphosphate (PIP2) than WT. Therefore, consistent with the inability of Kir2.1C122Y channels to bind directly to PIP2 in bioluminescence resonance energy transfer experiments, the PIP2 binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch-clamping the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing PIP2 concentrations. Conclusion: The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential to channel function. We demonstrated that ATS1 mutations that break disulfide bonds in the extracellular domain disrupt PIP2-dependent regulation, leading to channel dysfunction and life-threatening arrhythmias.

Intermediate molecular phenotypes to identify genetic markers of anthracycline-induced cardiotoxicity risk.

Cardiotoxicity due to anthracyclines (CDA) affects cancer patients, but we cannot predict who may suffer from this complication. CDA is a complex disease whose polygenic component is mainly unidentified. We propose that levels of intermediate molecular phenotypes in the myocardium associated with histopathological damage could explain CDA susceptibility; so that variants of genes encoding these intermediate molecular phenotypes could identify patients susceptible to this complication. A genetically heterogeneous cohort of mice generated by backcrossing (N = 165) was treated with doxorubicin and docetaxel. Cardiac histopathological damage was measured by fibrosis and cardiomyocyte size by an Ariol slide scanner. We determine intramyocardial levels of intermediate molecular phenotypes of CDA associated with histopathological damage and quantitative trait loci (ipQTLs) linked to them. These ipQTLs seem to contribute to the missing heritability of CDA because they improve the heritability explained by QTL directly linked to CDA (cda-QTLs) through genetic models. Genes encoding these molecular subphenotypes were evaluated as genetic markers of CDA in three cancer patient cohorts (N = 517) whose cardiac damage was quantified by echocardiography or Cardiac Magnetic Resonance. Many SNPs associated with CDA were found using genetic models. LASSO multivariate regression identified two risk score models, one for pediatric cancer patients and the other for women with breast cancer. Molecular intermediate phenotypes associated with heart damage can identify genetic markers of CDA risk, thereby allowing a more personalized patient management. A similar strategy could be applied to identify genetic markers of other complex trait diseases.

Molecular stratification of arrhythmogenic mechanisms in the Andersen Tawil syndrome.

Andersen-Tawil syndrome (ATS) is a rare inheritable disease associated with loss-of-function mutations in KCNJ2, the gene coding the strong inward rectifier potassium channel Kir2.1, which forms an essential membrane protein controlling cardiac excitability. ATS is usually marked by a triad of periodic paralysis, life-threatening cardiac arrhythmias and dysmorphic features, but its expression is variable and not all patients with a phenotype linked to ATS have a known genetic alteration. The mechanisms underlying this arrhythmogenic syndrome are poorly understood. Knowing such mechanisms would be essential to distinguish ATS from other channelopathies with overlapping phenotypes and to develop individualized therapies. For example, the recently suggested role of Kir2.1 as a countercurrent to sarcoplasmic calcium reuptake might explain the arrhythmogenic mechanisms of ATS and its overlap with catecholaminergic polymorphic ventricular tachycardia. Here we summarize current knowledge on the mechanisms of arrhythmias leading to sudden cardiac death in ATS. We first provide an overview of the syndrome and its pathophysiology, from the patient's bedside to the protein and discuss the role of essential regulators and interactors that could play a role in cases of ATS. The review highlights novel ideas related to some post-translational channel interactions with partner proteins that might help define the molecular bases of the arrhythmia phenotype. We then propose a new all-embracing classification of the currently known ATS loss-of-function mutations according to their position in the Kir2.1 channel structure and their functional implications. We also discuss specific ATS pathogenic variants, their clinical manifestations, and treatment stratification. The goal is to provide a deeper mechanistic understanding of the syndrome toward the development of novel targets and personalized treatment strategies.

MKK6 deficiency promotes cardiac dysfunction through MKK3-p38γ/δ-mTOR hyperactivation.

Stress-activated p38 kinases control a plethora of functions, and their dysregulation has been linked to the development of steatosis, obesity, immune disorders, and cancer. Therefore, they have been identified as potential targets for novel therapeutic strategies. There are four p38 family members (p38α, p38ß, p38γ, and p38δ) that are activated by MKK3 and MKK6. Here, we demonstrate that lack of MKK6 reduces the lifespan in mice. Longitudinal study of cardiac function in MKK6 KO mice showed that young mice develop cardiac hypertrophy which progresses to cardiac dilatation and fibrosis with age. Mechanistically, lack of MKK6 blunts p38α activation while causing MKK3-p38γ/δ hyperphosphorylation and increased mammalian target of rapamycin (mTOR) signaling, resulting in cardiac hypertrophy. Cardiac hypertrophy in MKK6 KO mice is reverted by knocking out either p38γ or p38δ or by inhibiting the mTOR pathway with rapamycin. In conclusion, we have identified a key role for the MKK3/6-p38γ/δ pathway in the development of cardiac hypertrophy, which has important implications for the clinical use of p38α inhibitors in the long-term treatment since they might result in cardiotoxicity.

The human heart can increase its size to supply more blood to the body's organs. This process, called hypertrophy, can happen during exercise or be caused by medical conditions, such as high blood pressure or inherited genetic diseases. If hypertrophy is continually driven by illness, this can cause the heart to fail and no longer be able to properly pump blood around the body. For hypertrophy to happen, several molecular changes occur in the cells responsible for contracting the heart, including activation of the p38 pathway. Within this pathway is a p38 enzyme as well as a series of other proteins which are sequentially turned on in response to stress, such as inflammatory molecules or mechanical forces that alter the cell's shape. There are different types of p38 enzyme which have been linked to other diseases, making them a promising target for drug development. However, clinical trials blocking individual members of the p38 family have had disappointing results. An alternative approach is to target other proteins involved in the p38 pathway, such as MKK6, but it is not known what effect this might have. To investigate, Romero-Becerra et al. genetically modified mice to not have any MKK6 protein. As a result, these mice had a shorter lifespan, with hypertrophy developing at a young age that led to heart problems. Romero-Becerra et al. used different mice models to understand why this happened, showing that a lack of MKK6 reduces the activity of a specific member of the p38 family called p38α. However, this blockage boosted a different branch of the pathway which involved two other p38 proteins, p38γ and p38δ. This, in turn, triggered another key pathway called mTOR which also promotes hypertrophy of the heart. These results suggest that drugs blocking MKK6 and p38α could lead to side effects that cause further harm to the heart. A more promising approach for treating hypertrophic heart conditions could be to inhibit p38γ and/or p38δ. However, before this can be fully explored, further work is needed to generate compounds that specifically target these proteins.

Distinct spectral dynamics of implanted cardiac defibrillator signals in spontaneous termination of polymorphic ventricular tachycardia and fibrillation in patients with electrical and structural diseases.

AIMS: To determine the spectral dynamics of early spontaneous polymorphic ventricular tachycardia and ventricular fibrillation (PVT/VF) in humans. METHODS AND RESULTS: Fifty-eight self-terminated and 173 shock-terminated episodes of spontaneously initiated PVT/VF recorded by Medtronic implanted cardiac defibrillators (ICDs) in 87 patients with various cardiac pathologies were analyzed by short fast Fourier transform of shifting segments to determine the dynamics of dominant frequency (DF) and regularity index (RI). The progression in the intensity of DF and RI accumulations further quantified the time course of spectral characteristics of the episodes. Episodes of self-terminated PVT/VF lasted 8.6 s [95% confidence interval (CI): 8.1-9.1] and shock-terminated lasted 13.9 s (13.6-14.3) (P < 0.001). Recordings from patients with primarily electrical pathologies displayed higher DF and RI values than those from patients with primarily structural pathologies (P < 0.05) independently of ventricular function or antiarrhythmic drug therapy. Regardless of the underlying pathology, the average DF and RI intensities were lower in self-terminated than shock-terminated episodes [DF: 3.67 (4.04-4.58) vs. 4.32 (3.46-3.93) Hz, P < 0.001; RI: 0.53 (0.48-0.56) vs. 0.63 (0.60-0.65), P < 0.001]. In a multivariate analysis controlled by the type of pathology and clinical variables, regularity remained an independent predictor of self-termination [hazard ratio: 0.954 (0.928-0.980)]. Receiver operating characteristic (ROC) curve analysis of DF and RI intensities demonstrated increased predictability for self-termination in time with 95% CI above the 0.5 cut-off limit at about t = 8.6 s and t = 6.95 s, respectively. CONCLUSION: Consistent with the notion that fast organized sources maintain PVT/VF in humans, reduction of frequency and regularity correlates with early self-termination. Our findings might help generate ICD methods aiming to reduce inappropriate shock deliveries.

SNTA1 gene rescues ion channel function and is antiarrhythmic in cardiomyocytes derived from induced pluripotent stem cells from muscular dystrophy patients.

Background: Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients. Methods: To test whether dystrophin mutations lead to defective cardiac NaV1.5-Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays. Results: Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers. Conclusions: We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5-Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5-Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias. Funding: Supported by National Institutes of Health R01 HL122352 grant; 'la Caixa' Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.

Tbx5 variants disrupt Nav1.5 function differently in patients diagnosed with Brugada or Long QT Syndrome.

AIMS: The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p. D111Y and p. F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with long QT (LQTS) and Brugada (BrS) syndrome. Here, we characterized the consequences of each variant to unravel the underlying disease mechanisms. METHODS AND RESULTS: We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans-expressing human wild-type (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca/calmodulin kinase IIδ (CaMKIIδ) and ßIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans-expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p. F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p. F206L markedly decreased INa in hiPSC-CM, HL-1 cells and mouse cardiomyocytes. The INa decrease in p. F206L trans-expressing mice translated into QRS widening and increased flecainide sensitivity. p. D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and ßIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p. D111Y trans-expressing mice. CONCLUSIONS: In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients.

Paclitaxel mitigates structural alterations and cardiac conduction system defects in a mouse model of Hutchinson-Gilford progeria syndrome.

AIMS: Hutchinson-Gilford progeria syndrome (HGPS) is an ultrarare laminopathy caused by expression of progerin, a lamin A variant, also present at low levels in non-HGPS individuals. HGPS patients age and die prematurely, predominantly from cardiovascular complications. Progerin-induced cardiac repolarization defects have been described previously, although the underlying mechanisms are unknown. METHODS AND RESULTS: We conducted studies in heart tissue from progerin-expressing LmnaG609G/G609G (G609G) mice, including microscopy, intracellular calcium dynamics, patch-clamping, in vivo magnetic resonance imaging, and electrocardiography. G609G mouse cardiomyocytes showed tubulin-cytoskeleton disorganization, t-tubular system disruption, sarcomere shortening, altered excitation-contraction coupling, and reductions in ventricular thickening and cardiac index. G609G mice exhibited severe bradycardia, and significant alterations of atrio-ventricular conduction and repolarization. Most importantly, 50% of G609G mice had altered heart rate variability, and sinoatrial block, both significant signs of premature cardiac aging. G609G cardiomyocytes had electrophysiological alterations, which resulted in an elevated action potential plateau and early afterdepolarization bursting, reflecting slower sodium current inactivation and long Ca+2 transient duration, which may also help explain the mild QT prolongation in some HGPS patients. Chronic treatment with low-dose paclitaxel ameliorated structural and functional alterations in G609G hearts. CONCLUSIONS: Our results demonstrate that tubulin-cytoskeleton disorganization in progerin-expressing cardiomyocytes causes structural, cardiac conduction, and excitation-contraction coupling defects, all of which can be partially corrected by chronic treatment with low dose paclitaxel.

Panoramic Endocardial Optical Mapping Demonstrates Serial Rotors Acceleration and Increasing Complexity of Activity During Onset of Cholinergic Atrial Fibrillation.

Background Activation during onset of atrial fibrillation is poorly understood. We aimed at developing a panoramic optical mapping system for the atria and test the hypothesis that sequential rotors underlie acceleration of atrial fibrillation during onset. Methods and Results Five sheep hearts were Langendorff perfused in the presence of 0.25 µmol/L carbachol. Novel optical system recorded activations simultaneously from the entire left and right atrial endocardial surfaces. Twenty sustained (>40 s) atrial fibrillation episodes were induced by a train and premature stimuli protocol. Movies obtained immediately (Initiation stage) and 30 s (Early Stabilization stage) after premature stimulus were analyzed. Serial rotor formation was observed in all sustained inductions and none in nonsustained inductions. In sustained episodes maximal dominant frequency increased from (mean±SD) 11.5±1.74 Hz during Initiation to 14.79±1.30 Hz at Early Stabilization (P<0.0001) and stabilized thereafter. At rotor sites, mean cycle length (CL) during 10 prerotor activations increased every cycle by 0.53% (P=0.0303) during Initiation and 0.34% (P=0.0003) during Early Stabilization. In contrast, CLs at rotor sites showed abrupt decreases after the rotors appearances by a mean of 9.65% (P<0.0001) during both stages. At Initiation, atria-wide accelerations and decelerations during rotors showed a net acceleration result whereby post-rotors atria-wide minimal CL (CLmin) were 95.5±6.8% of the prerotor CLmin (P=0.0042). In contrast, during Early Stabilization, there was no net acceleration in CLmin during accelerating rotors (prerotor=84.9±11.0% versus postrotor=85.8±10.8% of Initiation, P=0.4029). Levels of rotor drift distance and velocity correlated with atria-wide acceleration. Nonrotor phase singularity points did not accelerate atria-wide activation but multiplied during Initiation until Early Stabilization. Increasing number of singularity points, indicating increased complexity, correlated with atria-wide CLmin reduction (P<0.0001). Conclusions Novel panoramic optical mapping of the atria demonstrates shortening CL at rotor sites during cholinergic atrial fibrillation onset. Atrial fibrillation acceleration toward Early Stabilization correlates with the net result of atria-wide accelerations during drifting rotors activity.

Time-efficient three-dimensional transmural scar assessment provides relevant substrate characterization for ventricular tachycardia features and long-term recurrences in ischemic cardiomyopathy.

Delayed gadolinium-enhanced cardiac magnetic resonance (LGE-CMR) imaging requires novel and time-efficient approaches to characterize the myocardial substrate associated with ventricular arrhythmia in patients with ischemic cardiomyopathy. Using a translational approach in pigs and patients with established myocardial infarction, we tested and validated a novel 3D methodology to assess ventricular scar using custom transmural criteria and a semiautomatic approach to obtain transmural scar maps in ventricular models reconstructed from both 3D-acquired and 3D-upsampled-2D-acquired LGE-CMR images. The results showed that 3D-upsampled models from 2D LGE-CMR images provided a time-efficient alternative to 3D-acquired sequences to assess the myocardial substrate associated with ischemic cardiomyopathy. Scar assessment from 2D-LGE-CMR sequences using 3D-upsampled models was superior to conventional 2D assessment to identify scar sizes associated with the cycle length of spontaneous ventricular tachycardia episodes and long-term ventricular tachycardia recurrences after catheter ablation. This novel methodology may represent an efficient approach in clinical practice after manual or automatic segmentation of myocardial borders in a small number of conventional 2D LGE-CMR slices and automatic scar detection.

Mapping Technologies for Catheter Ablation of Atrial Fibrillation Beyond Pulmonary Vein Isolation.

Catheter ablation remains the most effective and relatively minimally invasive therapy for rhythm control in patients with AF. Ablation has consistently shown a reduction of arrhythmia-related symptoms and significant improvement in patients' quality of life compared with medical treatment. The ablation strategy relies on a well-established anatomical approach of effective pulmonary vein isolation. Additional anatomical targets have been reported with the aim of increasing procedure success in complex substrates. However, larger ablated areas with uncertainty of targeting relevant regions for AF initiation or maintenance are not exempt from the potential risk of complications and pro-arrhythmia. Recent developments in mapping tools and computational methods for advanced signal processing during AF have reported novel strategies to identify atrial regions associated with AF maintenance. These novel tools - although mainly limited to research series - represent a significant step forward towards the understanding of complex patterns of propagation during AF and the potential achievement of patient-tailored AF ablation strategies for the near future.

Novel approaches to mechanism-based atrial fibrillation ablation.

Modern cardiac electrophysiology has reported significant advances in the understanding of mechanisms underlying complex wave propagation patterns during atrial fibrillation (AF), although disagreements remain. One school of thought adheres to the long-held postulate that AF is the result of randomly propagating wavelets that wonder throughout the atria. Another school supports the notion that AF is deterministic in that it depends on a small number of high-frequency rotors generating three-dimensional scroll waves that propagate throughout the atria. The spiralling waves are thought to interact with anatomic and functional obstacles, leading to fragmentation and new wavelet formation associated with the irregular activation patterns documented on AF tracings. The deterministic hypothesis is consistent with demonstrable hierarchical gradients of activation frequency and AF termination on ablation at specific (non-random) atrial regions. During the last decade, data from realistic animal models and pilot clinical series have triggered a new era of novel methodologies to identify and ablate AF drivers outside the pulmonary veins. New generation electroanatomical mapping systems and multielectrode mapping catheters, complimented by powerful mathematical analyses, have generated the necessary platforms and tools for moving these approaches into clinical procedures. Recent clinical data using such platforms have provided encouraging evidence supporting the feasibility of targeting and effectively ablating driver regions in addition to pulmonary vein isolation in persistent AF. Here, we review state-of-the-art technologies and provide a comprehensive historical perspective, characterization, classification, and expected outcomes of current mechanism-based methods for AF ablation. We discuss also the challenges and expected future directions that scientists and clinicians will face in their efforts to understand AF dynamics and successfully implement any novel method into regular clinical practice.

Cardiac phenotype in familial partial lipodystrophy.

OBJECTIVES: LMNA variants have been previously associated with cardiac abnormalities independent of lipodystrophy. We aimed to assess cardiac impact of familial partial lipodystrophy (FPLD) to understand the role of laminopathy in cardiac manifestations. STUDY DESIGN: Retrospective cohort study. METHODS: Clinical data from 122 patients (age range: 13-77, 101 females) with FPLD were analysed. Mature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with an LMNA variant were studied as proof-of-concept for future studies. RESULTS: Subjects with LMNA variants had a higher prevalence of overall cardiac events than others. The likelihood of having an arrhythmia was significantly higher in patients with LMNA variants (OR: 3.77, 95% CI: 1.45-9.83). These patients were at higher risk for atrial fibrillation or flutter (OR: 5.78, 95% CI: 1.04-32.16). The time to the first arrhythmia was significantly shorter in the LMNA group, with a higher HR of 3.52 (95% CI: 1.34-9.27). Non-codon 482 LMNA variants were more likely to be associated with cardiac events (vs. 482 LMNA: OR: 4.74, 95% CI: 1.41-15.98 for arrhythmia; OR: 17.67, 95% CI: 2.45-127.68 for atrial fibrillation or flutter; OR: 5.71, 95% CI: 1.37-23.76 for conduction disease). LMNA mutant hiPSC-CMs showed a higher frequency of spontaneous activity and shorter action potential duration. Functional syncytia of hiPSC-CMs displayed several rhythm alterations such as early afterdepolarizations, spontaneous quiescence and spontaneous tachyarrhythmia, and significantly slower recovery in chronotropic changes induced by isoproterenol exposure. CONCLUSIONS: Our results highlight the need for vigilant cardiac monitoring in FPLD, especially in patients with LMNA variants who have an increased risk of developing cardiac arrhythmias. In addition, hiPSC-CMs can be studied to understand the basic mechanisms for the arrhythmias in patients with lipodystrophy to understand the impact of specific mutations.