Electrophysiologic characteristics of cells spanning the left ventricular wall of human heart: Evidence for presence of M cells

Objectives.The present work was designed to provide an initial characterization of M cells in the normal human heart.Background.Recent studies have uncovered a unique population of cells in the midmyocardial region of the canine ventricle. These cells, named M cells, were found to possess electrophysiologic features and a pharmacologic responsiveness different from those of other myocardial cells. Although well characterized in the dog, their presence or absence in the human heart is unknown.Methods.Standard microelectrode techniques were used to map slices of ventricular free wall obtained from normal human hearts (n = 4). Preparations were paced at cycle lengths ranging from 1 to 10 s.Results.We identified three cell subtypes: endocardial, subepicardial (M cells) and epicardial cells. The principal features differentiating M cells from the other cell subtypes were their longer action potential duration, more accentuated action potential duration rate relations and greater maximal rate of increase in action potential upstroke (Vmax). Our findings suggest that M cells represent ∼ 30% of the cellular mass of the left ventricular wall. Concordance between changes in their repolarization and changes in QTU interval provide support for the role of M cells in the generation of the electrocardiographic (ECG) U wave.Conclusions.This study provides evidence for the existence of M cells in the human heart that contribute to heterogeneity of repolarization within the ventricular wall. Our findings provide strong support for the hypothesis that M cells contribute importantly to the manifestation of the U wave on the ECG

KCNQ1 Antibodies for Immunotherapy of Long QT Syndrome Type 2

Background: Patients with long QT syndrome (LQTS) are predisposed to life-threatening arrhythmias. A delay in cardiac repolarization is characteristic of the disease. Pharmacotherapy, implantable cardioverter-defibrillators, and left cardiac sympathetic denervation are part of the current treatment options, but no targeted therapy for LQTS exists to date. Previous studies indicate that induced autoimmunity against the voltage-gated KCNQ1 K+ channels accelerates cardiac repolarization. Objectives: However, a causative relationship between KCNQ1 antibodies and the observed electrophysiological effects has never been demonstrated, and thus presents the aim of this study. Methods: The authors purified KCNQ1 antibodies and performed whole-cell patch clamp experiments as well as single-channel recordings on Chinese hamster ovary cells overexpressing IKs channels. The effect of purified KCNQ1 antibodies on human cardiomyocytes derived from induced pluripotent stem cells was then studied. Results: The study demonstrated that KCNQ1 antibodies underlie the previously observed increase in repolarizing IKs current. The antibodies shift the voltage dependence of activation and slow the deactivation of IKs. At the single-channel level, KCNQ1 antibodies increase the open time and probability of the channel. In models of LQTS type 2 (LQTS2) using human induced pluripotent stem cell-derived cardiomyocytes, KCNQ1 antibodies reverse the prolonged cardiac repolarization and abolish arrhythmic activities. Conclusions: Here, the authors provide the first direct evidence that KCNQ1 antibodies act as agonists on IKs channels. Moreover, KCNQ1 antibodies were able to restore alterations in cardiac repolarization and most importantly to suppress arrhythmias in LQTS2. KCNQ1 antibody therapy may thus present a novel promising therapeutic approach for LQTS2

Growing Teratoma Syndrome: An Asian Woman with Immature Teratoma of Left Ovary After Chemotherapy

International audienceAims: Loss-of-function of the cardiac sodium channel NaV1.5 is a common feature of Brugada syndrome. Arrhythmias arise preferentially from the right ventricle (RV) despite equivalent NaV1.5 downregulation in the left ventricle (LV). The reasons for increased RV sensitivity to NaV1.5 loss-of-function mutations remain unclear. Because ventricular electrical activation occurs predominantly in the transmural axis, we compare RV and LV transmural electrophysiology to determine the underlying cause of the asymmetrical conduction abnormalities in Scn5a haploinsufficient mice (Scn5a+/-). Methods and results: Optical mapping and two-photon microscopy in isolated-perfused mouse hearts demonstrated equivalent depression of transmural conduction velocity (CV) in the LV and RV of Scn5a+/- vs. wild-type littermates. Only RV transmural conduction was further impaired when challenged with increased pacing frequencies. Epicardial dispersion of activation and beat-to-beat variation in activation time were increased only in the RV of Scn5a+/- hearts. Analysis of confocal and histological images revealed larger intramural clefts between cardiomyocyte layers in the RV vs. LV, independent of genotype. Acute sodium current inhibition in wild type hearts using tetrodotoxin reproduced beat-to-beat activation variability and frequency-dependent CV slowing in the RV only, with the LV unaffected. The influence of clefts on conduction was examined using a two-dimensional monodomain computational model. When peak sodium channel conductance was reduced to 50% of normal the presence of clefts between cardiomyocyte layers reproduced the activation variability and conduction phenotype observed experimentally. Conclusions: Normal structural heterogeneities present in the RV are responsible for increased vulnerability to conduction slowing in the presence of reduced sodium channel function. Heterogeneous conduction slowing seen in the RV will predispose to functional block and the initiation of re-entrant ventricular arrhythmias

C-terminal phosphorylation of NaV1.5 impairs FGF13-dependent regulation of channel inactivation

International audienceVoltage-gated Na(+) (NaV) channels are key regulators of myocardial excitability, and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent alterations in NaV1.5 channel inactivation are emerging as a critical determinant of arrhythmias in heart failure. However, the global native phosphorylation pattern of NaV1.5 subunits associated with these arrhythmogenic disorders and the associated channel regulatory defects remain unknown. Here, we undertook phosphoproteomic analyses to identify and quantify in situ the phosphorylation sites in the NaV1.5 proteins purified from adult WT and failing CaMKIIδc-overexpressing (CaMKIIδc-Tg) mouse ventricles. Of 19 native NaV1.5 phosphorylation sites identified, two C-terminal phosphoserines at positions 1938 and 1989 showed increased phosphorylation in the CaMKIIδc-Tg compared with the WT ventricles. We then tested the hypothesis that phosphorylation at these two sites impairs fibroblast growth factor 13 (FGF13)-dependent regulation of NaV1.5 channel inactivation. Whole-cell voltage-clamp analyses in HEK293 cells demonstrated that FGF13 increases NaV1.5 channel availability and decreases late Na(+) current, two effects that were abrogated with NaV1.5 mutants mimicking phosphorylation at both sites. Additional co-immunoprecipitation experiments revealed that FGF13 potentiates the binding of calmodulin to NaV1.5 and that phosphomimetic mutations at both sites decrease the interaction of FGF13 and, consequently, of calmodulin with NaV1.5. Together, we have identified two novel native phosphorylation sites in the C terminus of NaV1.5 that impair FGF13-dependent regulation of channel inactivation and may contribute to CaMKIIδc-dependent arrhythmogenic disorders in failing hearts

0134 : Using cardiomyocytes differentiated from urine-derived hiPSCs to recapitulate electrophysiological characteristics of LQT2 syndrome

RationaleHuman genetically inherited cardiac diseases have mainly been studied in heterologous systems or animal models, independently of the patients’ genetic background. Because sources for human cardiomyocytes are extremely limited, the use of urine samples to derive cardiomyocytes would be a non-invasive method to identify cardiac dysfunctions that lead to pathologies within the patients’ specific genetic background.ObjectiveCardiomyocytes differentiated from urine-derived pluripotent stem cells (UhiPS-CMs) were obtained from a patient with long QT syndrome and a mutation in hERG KCNH2 gene (p. A561P), and were characterized.Methods and ResultsCells obtained from urine samples from the A561P patient and his asymptomatic mother carrying no hERG mutation were reprogrammed using the episomal-based method. UhiPS cells were then differentiated into cardiomyocytes using a modified matrix sandwich method. UhiPS-CMs showed proper expression of ventricular cytoskeletal proteins and ion channels. They were electrically functional, with nodal-, atrial- and ventricular-like action potentials (APs) recorded using both high-throughput CellOptiq and patch-clamp techniques. Application of ajmaline, 4-aminopyridine, nifedipine, chromanol 293B or E-4031 to the UhiPS-CMs confirmed that INa, Ito, ICa, IKs and IKr currents, respectively, contributed to the APs. Comparing hERG expression from the patient's UhiPS-CMs to the mother's UhiPS-CMs showed that the mutation led to a trafficking defect that resulted in a reduced IKr current. This phenotype led to APs prolongation that sometimes resulted in arrhythmias (early afterdepolarizations).ConclusionUrine-derived pluripotent stem cells from patients carrying ion channels mutations can be used as novel models to differentiate functional cardiomyocytes that recapitulate cardiac arrhythmia phenotypes

Intracardiac electrophysiology to characterize susceptibility to ventricular arrhythmias in murine models

Introduction: Sudden cardiac death (SCD) and ventricular fibrillation are rare but severe complications of many cardiovascular diseases and represent a major health issue worldwide. Although the primary causes are often acute or chronic coronary diseases, genetic conditions, such as inherited channelopathies or non-ischemic cardiomyopathies are leading causes of SCD among the young. However, relevant experimental models to study the underlying mechanisms of arrhythmias and develop new therapies are still needed. The number of genetically engineered mouse models with cardiac phenotype is growing, making electrophysiological studies in mice essential tools to study arrhythmogenicity and arrhythmia mechanisms and to test novel treatments. Recently, intracardiac catheterization via the jugular vein was described to induce and record ventricular arrhythmias in living anesthetized mice. Several strategies have been reported, developed in healthy wild-type animals and based on aggressive right ventricular stimulation.Methods: Here, we report a protocol based on programmed electrical stimulation (PES) performed in clinical practice in patients with cardiac rhythm disorders, adapted to two transgenic mice models of arrhythmia - Brugada syndrome and cardiolaminopathy.Results: We show that this progressive protocol, based on a limited number of right ventricular extrastimuli, enables to reveal different rhythmic phenotypes between control and diseased mice. In this study, we provide detailed information on PES in mice, including catheter positioning, stimulation protocols, intracardiac and surface ECG interpretation and we reveal a higher susceptibility of two mouse lines to experience triggered ventricular arrhythmias, when compared to control mice.Discussion: Overall, this technique allows to characterize arrhythmias and provides results in phenotyping 2 arrhythmogenic-disease murine models

Variable Nav1.5 Protein Expression from the Wild-Type Allele Correlates with the Penetrance of Cardiac Conduction Disease in the Scn5a+/− Mouse Model

BACKGROUND: Loss-of-function mutations in SCN5A, the gene encoding Na(v)1.5 Na+ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a(+/-) mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A. METHODOLOGY/PRINCIPAL FINDINGS: Based on ECG, 10-week-old Scn5a(+/-) mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a(+/-) mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A-mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a(+/-) mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a(+/-) mice had similar Na(v)1.5 mRNA but higher Na(v)1.5 protein expression, and moderately larger I(Na) current than severely affected Scn5a(+/-) mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a(+/-) mice than in mildly affected ones. CONCLUSIONS: Scn5a(+/-) mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a(+/-) mice, phenotype severity correlates with wild-type Na(v)1.5 protein expression