Jacques M.T. de Bakker

Right ventricular fibrosis and conduction delay in a patient with clinical signs of Brugada syndrome : a combined electrophysiological, genetic, histopathologic, and computational study

Background— The mechanism of ECG changes and arrhythmogenesis in Brugada syndrome (BS) patients is unknown. Methods and Results— A BS patient without clinically detected cardiac structural abnormalities underwent cardiac transplantation for intolerable numbers of implantable cardioverter/defibrillator discharges. The patient’s explanted heart was studied electrophysiologically and histopathologically. Whole-cell currents were measured in HEK293 cells expressing wild-type or mutated sodium channels from the patient. The right ventricular outflow tract (RVOT) endocardium showed activation slowing and was the origin of ventricular fibrillation without a transmural repolarization gradient. Conduction restitution was abnormal in the RVOT but normal in the left ventricle. Right ventricular hypertrophy and fibrosis with epicardial fatty infiltration were present. HEK293 cells expressing a G1935S mutation in the gene encoding the cardiac sodium channel exhibited enhanced slow inactivation compared with wild-type channels. Computer simulations demonstrated that conduction slowing in the RVOT might have been the cause of the ECG changes. Conclusions— In this patient with BS, conduction slowing based on interstitial fibrosis, but not transmural repolarization differences, caused the ECG signs and was the origin of ventricular fibrillation.

Electrical Conduction in Canine Pulmonary Veins: Electrophysiological and Anatomic Correlation

Background—Paroxysmal atrial fibrillation in patients is often initiated by foci in the pulmonary veins. The mechanism of these initiating arrhythmias is unknown. The aim of this study was to determine electrophysiological characteristics of canine pulmonary veins that may predispose to initiating arrhythmias. Methods and Results—Extracellular recordings were obtained from the luminal side of 9 pulmonary veins in 6 Langendorff-perfused dog hearts after the veins were incised from the severed end to the ostium. Pulmonary veins were paced at the distal end, the ostium, and an intermediate site. During basic and premature stimulation, extracellular electrical activity was recorded with a grid electrode that harbored 247 electrode terminals. In 4 hearts, intracellular electrograms were recorded with microelectrodes. Myocyte arrangement immediately beneath the venous walls was determined by histological analysis in 3 hearts. Extracellular mapping revealed slow and complex conduction in all pulmonary veins. Activation delay after premature stimulation could be as long as 96 ms over a distance of 3 mm. Action potential duration was shorter at the distal end of the veins than at the orifice. No evidence for automaticity or triggered activity was found. Histological investigation revealed complex arrangements of myocardial fibers that often showed abrupt changes in fiber direction and short fibers arranged in mixed direction. Conclusions—Zones of activation delay were observed in canine pulmonary veins and correlated with abrupt changes in fascicle orientation. This architecture of muscular sleeves in the pulmonary veins may facilitate reentry and arrhythmias associated with ectopic activity.

Slow Conduction and Enhanced Anisotropy Increase the Propensity for Ventricular Tachyarrhythmias in Adult Mice With Induced Deletion of Connexin43

Background—Connexin 43 (Cx43) is a major determinant of conduction in the ventricular working myocardium of mammals. We investigated the effect of decreased Cx43 expression on conduction velocity and arrhythmogenesis using adult mice with inducible deletion of Cx43. Methods and Results—Cx43Cre-ER(T)/+ mice, in which 1 coding region of the Cx43 gene was replaced by Cre-ER(T), were mated to Cx43fl/fl mice, generating Cx43Cre-ER(T)/fl mice. Application of 4-hydroxytamoxifen (4-OHT) induced Cre-ER(T)–mediated deletion of the floxed Cx43 allele. Epicardial ventricular mapping using a 13×19 multiterminal electrode grid (300-&mgr;m spacing) was performed on Langendorff-perfused hearts from Cx43fl/fl plus carrier (n=10), Cx43fl/fl plus 4-OHT (n=10), Cx43 Cre-ER(T)/fl plus carrier (n=9), and Cx43Cre-ER(T)/fl plus 4-OHT (n=10). Cx43 protein amount in group 3 hearts was decreased by ≈50% compared with group 1. 4-OHT did not affect cardiac protein amounts in group 2 but decreased Cx43 expression up to 95% in group 4 compared with group 3. Epicardial activation of both left ventricle (LV) and right ventricle (RV) during sinus rhythm was similar in all groups. Conduction velocity (CV) changed only in group 4 animals. For RV (LV), longitudinal CV decreased from 38 (35) to 31.6 (33.6) and transverse CV from 24.4 (16.8) to 10.1 (11.3) cm/s. Dispersion of conduction in RV (LV) was increased by 91% (38%). Programmed stimulation resulted in ventricular arrhythmias in group 4 (7 of 10 mice) but never in groups 1 through 3. Conclusions—Heterozygous expression of Cx43 did not affect ventricular conduction velocity. Up to 95% decrease of Cx43 protein in 4-OHT–treated Cx43Cre-ER(T)/fl mice reduced conduction velocity and increased dispersion of conduction and propensity for ventricular arrhythmias.

Mouse model of SCN5A-linked hereditary Lenegre's disease - Age-related conduction slowing and myocardial fibrosis

Background—We have previously linked hereditary progressive cardiac conduction defect (hereditary Lenègre’s disease) to a loss-of-function mutation in the gene encoding the main cardiac Na+ channel, SCN5A. In the present study, we investigated heterozygous Scn5a-knockout mice (Scn5a+/− mice) as a model for hereditary Lenègre’s disease. Methods and Results—In Scn5a+/− mice, surface ECG recordings showed age-related lengthening of the P-wave and PR- and QRS-interval duration, coinciding with previous observations in patients with Lenègre’s disease. Old but not young Scn5a+/− mice showed extensive fibrosis of their ventricular myocardium, a feature not seen in wild-type animals. In old Scn5a+/− mice, fibrosis was accompanied by heterogeneous expression of connexin 43 and upregulation of hypertrophic markers, including &bgr;-MHC and skeletal &agr;-actin. Global connexin 43 expression as assessed with Western blots was similar to wild-type mice. Decreased connexin 40 expression was seen in the atria. Using pangenomic microarrays and real-time PCR, we identified in Scn5a+/− mice an age-related upregulation of genes encoding Atf3 and Egr1 transcription factors. Echocardiography and hemodynamic investigations demonstrated conserved cardiac function with aging and lack of ventricular hypertrophy. Conclusions—We conclude that Scn5a+/− mice convincingly recapitulate the Lenègre’s disease phenotype, including progressive impairment with aging of atrial and ventricular conduction associated with myocardial rearrangements and fibrosis. Our work provides the first demonstration that a monogenic ion channel defect can progressively lead to myocardial structural anomalies.

Overlap Syndrome of Cardiac Sodium Channel Disease in Mice Carrying the Equivalent Mutation of Human SCN5A-1795insD

Background— Patients carrying the cardiac sodium channel (SCN5A) mutation 1795insD show sudden nocturnal death and signs of multiple arrhythmia syndromes including bradycardia, conduction delay, QT prolongation, and right precordial ST-elevation. We investigated the electrophysiological characteristics of a transgenic model of the murine equivalent mutation 1798insD. Methods and Results— On 24-hour continuous telemetry and surface ECG recordings, Scn5a1798insD/+ heterozygous mice showed significantly lower heart rates, more bradycardic episodes (pauses ≥500 ms), and increased PQ interval, QRS duration, and QTc interval compared with wild-type mice. The sodium channel blocker flecainide induced marked sinus bradycardia and/or sinus arrest in the majority of Scn5a1798insD/+ mice, but not in wild-type mice. Epicardial mapping using a multielectrode grid on excised, Langendorff-perfused hearts showed preferential conduction slowing in the right ventricle of Scn5a1798insD/+ hearts. On whole-cell patch-clamp analysis, ventricular myocytes isolated from Scn5a1798insD/+ hearts displayed action potential prolongation, a 39% reduction in peak sodium current density and a similar reduction in action potential upstroke velocity. Scn5a1798insD/+ myocytes displayed a slower time course of sodium current decay without significant differences in voltage-dependence of activation and steady-state inactivation, slow inactivation, or recovery from inactivation. Furthermore, Scn5a1798insD/+ myocytes showed a larger tetrodotoxin-sensitive persistent inward current compared with wild-type myocytes. Conclusions— Mice carrying the murine equivalent of the SCN5A-1795insD mutation display bradycardia, right ventricular conduction slowing, and QT prolongation, similar to the human phenotype. These results demonstrate that the presence of a single SCN5A mutation is indeed sufficient to cause an overlap syndrome of cardiac sodium channel disease.

Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Pathogenic Desmosome Mutations in Index-Patients Predict Outcome of Family Screening: Dutch Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy Genotype-Phenotype Follow-Up Study

Background— Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an autosomal dominant inherited disease with incomplete penetrance and variable expression. Causative mutations in genes encoding 5 desmosomal proteins are found in ≈50% of ARVD/C index patients. Previous genotype-phenotype relation studies involved mainly overt ARVD/C index patients, so follow-up data on relatives are scarce. Methods and Results— One hundred forty-nine ARVD/C index patients (111 male patients; age, 49±13 years) according to 2010 Task Force criteria and 302 relatives from 93 families (282 asymptomatic; 135 male patients; age, 44±13 years) were clinically and genetically characterized. DNA analysis comprised sequencing of plakophilin-2 ( PKP2 ), desmocollin-2, desmoglein-2, desmoplakin, and plakoglobin and multiplex ligation-dependent probe amplification to identify large deletions in PKP2. Pathogenic mutations were found in 87 index patients (58%), mainly truncating PKP2 mutations, including 3 cases with multiple mutations. Multiplex ligation-dependent probe amplification revealed 3 PKP2 exon deletions. ARVD/C was diagnosed in 31% of initially asymptomatic mutation-carrying relatives and 5% of initially asymptomatic relatives of index patients without mutation. Prolonged terminal activation duration was observed more than negative T waves in V1 to V3, especially in mutation-carrying relatives <20 years of age. In 45% of screened families, ≥1 affected relatives were identified (90% with mutations). Conclusions— Pathogenic desmosomal gene mutations, mainly truncating PKP2 mutations, underlie ARVD/C in the majority (58%) of Dutch index patients and even 90% of familial cases. Additional multiplex ligation-dependent probe amplification analysis contributed to discovering pathogenic mutations underlying ARVD/C. Discovering pathogenic mutations in index patients enables those relatives who have a 6-fold increased risk of ARVD/C diagnosis to be identified. Prolonged terminal activation duration seems to be a first sign of ARVD/C in young asymptomatic relatives. # Clinical Perspective {#article-title-38}

Thoracoscopic Video-Assisted Pulmonary Vein Antrum Isolation, Ganglionated Plexus Ablation and Periprocedural Confirmation of Ablation Lesions. First Results of a Hybrid Surgical-Electrophysiological Approach for Atrial Fibrillation

Background—Thoracoscopic pulmonary vein isolation (PVI) and ganglionated plexus ablation is a novel approach in the treatment of atrial fibrillation (AF). We hypothesize that meticulous electrophysiological confirmation of PVI results in fewer recurrences of AF during follow-up. Methods and Results—Surgery was performed through 3 ports bilaterally. Ganglionated plexi were localized and subsequently ablated. PVI was performed and entry and exit block was confirmed. Additional left atrial ablation lines were created and conduction block verified in patients with nonparoxysmal AF. The left atrial appendage was removed. Freedom of AF was assessed by ECGs and Holter monitoring every 3 months or during symptoms of arrhythmia. Antiarrhythmic drugs were discontinued after 3 months and oral anticoagulants were discontinued according to the guidelines. Thirty-one patients were treated (16 paroxysmal AF, 13 persistent AF, 2 long-standing persistent AF). Thirteen patients with nonparoxysmal received additional left atrial ablation lines. After 1 year, 19 of 22 patients (86%) had no recurrences of AF, atrial flutter, or atrial tachycardia and were not using antiarrhythmic drugs (11/12 paroxysmal, 7/9 persistent, and 1/1 long-standing persistent). Three patients had a sternotomy because of uncontrolled bleeding during thoracoscopic surgery. Four adverse events were 1 hemothorax, 1 pneumothorax, and 2 pneumonia. No thromboembolic complications or mortality occurred. Conclusions—Thoracoscopic surgery with PVI and ganglionated plexus ablation for AF is a safe and successful procedure with a single procedure success rate of 86% at 1 year. Electrophysiological guided thorough PVI and additional left atrial ablation line creation presumably contributes in achieving a high success rate in the surgical treatment of AF.

Impaired Impulse Propagation in Scn5a-Knockout Mice Combined Contribution of Excitability, Connexin Expression, and Tissue Architecture in Relation to Aging

Background—The SCN5A sodium channel is a major determinant for cardiac impulse propagation. We used epicardial mapping of the atria, ventricles, and septae to investigate conduction velocity (CV) in Scn5a heterozygous young and old mice. Methods and Results—Mice were divided into 4 groups: (1) young (3 to 4 months) wild-type littermates (WT); (2) young heterozygous Scn5a-knockout mice (HZ); (3) old (12 to 17 months) WT; and (4) old HZ. In young HZ hearts, CV in the right but not the left ventricle was reduced in agreement with a rightward rotation in the QRS axes; fibrosis was virtually absent in both ventricles, and the pattern of connexin43 (Cx43) expression was similar to that of WT mice. In old WT animals, the right ventricle transversal CV was slightly reduced and was associated with interstitial fibrosis. In old HZ hearts, right and left ventricle CVs were severely reduced both in the transversal and longitudinal direction; multiple areas of severe reactive fibrosis invaded the myocardium, accompanied by markedly altered Cx43 expression. The right and left bundle-branch CVs were comparable to those of WT animals. The atria showed only mild fibrosis, with heterogeneously disturbed Cx40 and Cx43 expression. Conclusions—A 50% reduction in Scn5a expression alone or age-related interstitial fibrosis only slightly affects conduction. In aged HZ mice, reduced Scn5a expression is accompanied by the presence of reactive fibrosis and disarrangement of gap junctions, which results in profound conduction impairment.

Impaired Conduction in the Bundle Branches of Mouse Hearts Lacking the Gap Junction Protein Connexin40

Background—Connexin (Cx)40 and Cx45 are the major protein subunits of gap junction channels in the conduction system of mammals. To determine the role of Cx40, we correlated cardiac activation with C onnexin distribution in normal and Cx40-deficient mice hearts. Methods and Results—Epicardial and septal activation was recorded in Langendorff-perfused adult mice hearts with a 247-point compound electrode (interelectrode distance, 0.3 mm). After electrophysiological measurements, hearts were prepared for immunohistochemistry and histology to determine C onnexin distribution and fibrosis. In both wild-type and Cx40-deficient animals, epicardial activation patterns were similar. The right and left ventricular septum was invariably activated from base to apex. Histology revealed a continuity of myocytes from the common bundle to the septal myocardium. Within this continuity, colocalization was found of Cx43 and Cx45 but not of Cx40 and Cx43. Both animals showed similar His-bundle activation. In Cx40-deficient mice, the proximal bundle branches expressed Cx45 only. The absence of Cx40 in the proximal bundles correlated with right bundle-branch block. Conduction in the left bundle branch was impaired as compared with wild-type animals. Conclusions—Our data show that (1) in mice, a continuity exists between the common bundle and the septum, and (2) Cx40 deficiency results in right bundle-branch block and impaired left bundle-branch conduction.

Fractionated electrograms in dilated cardiomyopathy: Origin and relation to abnormal conduction

OBJECTIVES We sought to investigate the origin of the fractionated electrogram and its relations to abnormal conduction in cardiomyopathic myocardium. BACKGROUND Patients with dilated cardiomyopathy have a high incidence of ventricular tachycardias. Electrograms recorded in these patients are often fractionated. METHODS High resolution mapping (200-microM interelectrode distance) of the electrical activity was carried out in 11 superfused papillary muscles and 6 trabeculae from 7 patients who underwent heart transplantation because of dilated cardiomyopathy. Similar measurements were taken in four papillary muscles from dog hearts in which electrical barriers had been artificially made. Ten human preparations were studied histologically. RESULTS All preparations revealed sites with fractionated electrograms. In three human preparations, activation patterns showed a discernible line of activation block running parallel to the fiber direction. Fractionated electrograms were recorded at sites contiguous to the line of block. In five preparations, fractionated electrograms were recorded at sites where lines of block were not identified. In these preparations, electrical barriers consisted of short stretches of fibrous tissue. In the remaining nine preparations, fractionated electrograms were recorded, both from sites contiguous to distinct obstacles and sites without evidence of a barrier. CONCLUSIONS Our observations showed that fractionated electrograms recorded in myocardium damaged by cardiomyopathy were due to both distinct, long strands and short stretches of fibrous tissue. Delayed conduction was caused by curvation of activation around the distinct lines of block and by the wavy course of activation between the short barriers. The latter reflects extreme nonuniform anisotropy.