Carol Ann Remme

Genotype-phenotype relationship in Brugada syndrome: electrocardiographic features differentiate SCN5A-related patients from non–SCN5A-related patients☆

OBJECTIVES We have tested whether a genotype-phenotype relationship exists in Brugada syndrome (BS) by trying to distinguish BS patients with (carriers) and those without (non-carriers) a mutation in the gene encoding the cardiac sodium channel (SCN5A) using clinical parameters. BACKGROUND Brugada syndrome is an inherited cardiac disease characterized by a varying degree of ST-segment elevation in the right precordial leads and (non)specific conduction disorders. In a minority of patients, SCN5A mutations can be found. Genetic heterogeneity has been demonstrated, but other causally related genes await identification. If a genotype-phenotype relationship exists, this might facilitate screening. METHODS In a multi-center study, we have collected data on demographics, clinical history, family history, electrocardiogram (ECG) parameters, His to ventricle interval (HV), and ECG parameters after pharmacologic challenge with I(Na) blocking drugs for BS patients with (n = 23), or those without (n = 54), an identified SCN5A mutation. RESULTS No differences were found in demographics, clinical history, or family history. Carriers had a significantly longer PQ interval on the baseline ECG and a significantly longer HV time. A PQ interval of > or =210 ms and an HV interval > or =60 ms seem to be predictive for the presence of an SCN5A mutation. After I(Na) blocking drugs, carriers had significantly longer PQ and QRS intervals and more increase in QRS duration. CONCLUSIONS We observed significantly longer conduction intervals on baseline ECG in patients with established SCN5A mutations (PQ and HV interval and, upon class I drugs, more QRS increase). These results concur with the observed loss of function of mutated BS-related sodium channels. Brugada syndrome patients with, and those without, an SCN5A mutation can be differentiated by phenotypical differences.

Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death

Brugada syndrome is a rare cardiac arrhythmia disorder, causally related to SCN5A mutations in around 20% of cases. Through a genome-wide association study of 312 individuals with Brugada syndrome and 1,115 controls, we detected 2 significant association signals at the SCN10A locus (rs10428132) and near the HEY2 gene (rs9388451). Independent replication confirmed both signals (meta-analyses: rs10428132, P = 1.0 × 10−68; rs9388451, P = 5.1 × 10−17) and identified one additional signal in SCN5A (at 3p21; rs11708996, P = 1.0 × 10−14). The cumulative effect of the three loci on disease susceptibility was unexpectedly large (Ptrend = 6.1 × 10−81). The association signals at SCN5A-SCN10A demonstrate that genetic polymorphisms modulating cardiac conduction can also influence susceptibility to cardiac arrhythmia. The implication of association with HEY2, supported by new evidence that Hey2 regulates cardiac electrical activity, shows that Brugada syndrome may originate from altered transcriptional programming during cardiac development. Altogether, our findings indicate that common genetic variation can have a strong impact on the predisposition to rare diseases.

Cardiomyocytes Derived From Pluripotent Stem Cells Recapitulate Electrophysiological Characteristics of an Overlap Syndrome of Cardiac Sodium Channel Disease

Background— Pluripotent stem cells (PSCs) offer a new paradigm for modeling genetic cardiac diseases, but it is unclear whether mouse and human PSCs can truly model both gain- and loss-of-function genetic disorders affecting the Na+ current (INa) because of the immaturity of the PSC-derived cardiomyocytes. To address this issue, we generated multiple PSC lines containing a Na+ channel mutation causing a cardiac Na+ channel overlap syndrome. Method and Results— Induced PSC (iPSC) lines were generated from mice carrying the Scn5a1798insD/+ (Scn5a-het) mutation. These mouse iPSCs, along with wild-type mouse iPSCs, were compared with the targeted mouse embryonic stem cell line used to generate the mutant mice and with the wild-type mouse embryonic stem cell line. Patch-clamp experiments showed that the Scn5a-het cardiomyocytes had a significant decrease in INa density and a larger persistent INa compared with Scn5a-wt cardiomyocytes. Action potential measurements showed a reduced upstroke velocity and longer action potential duration in Scn5a-het myocytes. These characteristics recapitulated findings from primary cardiomyocytes isolated directly from adult Scn5a-het mice. Finally, iPSCs were generated from a patient with the equivalent SCN5A1795insD/+ mutation. Patch-clamp measurements on the derivative cardiomyocytes revealed changes similar to those in the mouse PSC-derived cardiomyocytes. Conclusion— Here, we demonstrate that both embryonic stem cell- and iPSC-derived cardiomyocytes can recapitulate the characteristics of a combined gain- and loss-of-function Na+ channel mutation and that the electrophysiological immaturity of PSC-derived cardiomyocytes does not preclude their use as an accurate model for cardiac Na+ channel disease.

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.

Intercalated disc abnormalities, reduced Na+ current density, and conduction slowing in desmoglein-2 mutant mice prior to cardiomyopathic changes

AIMS Mutations in genes encoding desmosomal proteins have been implicated in the pathogenesis of arrhythmogenic right ventricular cardiomyopathy (ARVC). However, the consequences of these mutations in early disease stages are unknown. We investigated whether mutation-induced intercalated disc remodelling impacts on electrophysiological properties before the onset of cell death and replacement fibrosis. METHODS AND RESULTS Transgenic mice with cardiac overexpression of mutant Desmoglein2 (Dsg2) Dsg2-N271S (Tg-NS/L) were studied before and after the onset of cell death and replacement fibrosis. Mice with cardiac overexpression of wild-type Dsg2 and wild-type mice served as controls. Assessment by electron microscopy established that intercellular space widening at the desmosomes/adherens junctions occurred in Tg-NS/L mice before the onset of necrosis and fibrosis. At this stage, epicardial mapping in Langendorff-perfused hearts demonstrated prolonged ventricular activation time, reduced longitudinal and transversal conduction velocities, and increased arrhythmia inducibility. A reduced action potential (AP) upstroke velocity due to a lower Na(+) current density was also observed at this stage of the disease. Furthermore, co-immunoprecipitation demonstrated an in vivo interaction between Dsg2 and the Na(+) channel protein Na(V)1.5. CONCLUSION Intercellular space widening at the level of the intercalated disc (desmosomes/adherens junctions) and a concomitant reduction in AP upstroke velocity as a consequence of lower Na(+) current density lead to slowed conduction and increased arrhythmia susceptibility at disease stages preceding the onset of necrosis and replacement fibrosis. The demonstration of an in vivo interaction between Dsg2 and Na(V)1.5 provides a molecular pathway for the observed electrical disturbances during the early ARVC stages.

Cardiac sodium channelopathy associated with SCN5A mutations: electrophysiological, molecular and genetic aspects

Abstract  Over the last two decades, an increasing number of SCN5A mutations have been described in patients with long QT syndrome type 3 (LQT3), Brugada syndrome, (progressive) conduction disease, sick sinus syndrome, atrial standstill, atrial fibrillation, dilated cardiomyopathy, and sudden infant death syndrome (SIDS). Combined genetic, electrophysiological and molecular studies have provided insight into the dysfunction and dysregulation of the cardiac sodium channel in the setting of SCN5A mutations identified in patients with these inherited arrhythmia syndromes. However, risk stratification and patient management is hindered by the reduced penetrance and variable disease expressivity in sodium channelopathies. Furthermore, various SCN5A‐related arrhythmia syndromes are known to display mixed phenotypes known as cardiac sodium channel overlap syndromes. Determinants of variable disease expressivity, including genetic background and environmental factors, are suspected but still largely unknown. Moreover, it has become increasingly clear that sodium channel function and regulation is more complicated than previously assumed, and the sodium channel may play additional, as of yet unrecognized, roles in cardiac structure and function. Development of cardiac structural abnormalities secondary to SCN5A mutations has been reported, but the clinical relevance and underlying mechanisms are unclear. Increased insight into these issues would enable a major next step in research related to cardiac sodium channel disease, ultimately enabling improved diagnosis, risk stratification and treatment strategies.

Functional NaV1.8 Channels in Intracardiac Neurons The Link Between SCN10A and Cardiac Electrophysiology

Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A/Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 &mgr;mol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A/NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity.Rationale: The SCN10A gene encodes the neuronal sodium channel isoform NaV1.8. Several recent genome-wide association studies have linked SCN10A to PR interval and QRS duration, strongly suggesting an as-yet unknown role for NaV1.8 in cardiac electrophysiology. Objective: To demonstrate the functional presence of SCN10A /Nav1.8 in intracardiac neurons of the mouse heart. Methods and Results: Immunohistochemistry on mouse tissue sections showed intense NaV1.8 labeling in dorsal root ganglia and intracardiac ganglia and only modest NaV1.8 expression within the myocardium. Immunocytochemistry further revealed substantial NaV1.8 staining in isolated neurons from murine intracardiac ganglia but no NaV1.8 expression in isolated ventricular myocytes. Patch-clamp studies demonstrated that the NaV1.8 blocker A-803467 (0.5–2 μmol/L) had no effect on either mean sodium current (INa) density or INa gating kinetics in isolated myocytes but significantly reduced INa density in intracardiac neurons. Furthermore, A-803467 accelerated the slow component of current decay and shifted voltage dependence of inactivation toward more negative voltages, as expected for blockade of NaV1.8-based INa. In line with these findings, A-803467 did not affect cardiomyocyte action potential upstroke velocity but markedly reduced action potential firing frequency in intracardiac neurons, confirming a functional role for NaV1.8 in cardiac neural activity. Conclusions: Our findings demonstrate the functional presence of SCN10A /NaV1.8 in intracardiac neurons, indicating a novel role for this neuronal sodium channel in regulation of cardiac electric activity. # Novelty and Significance {#article-title-33}

Right Ventricular Failure Following Chronic Pressure Overload Is Associated With Reduction in Left Ventricular Mass : Evidence for Atrophic Remodeling

OBJECTIVES We sought to study whether patients with right ventricular failure (RVF) secondary to chronic thromboembolic pulmonary hypertension (CTEPH) have reduced left ventricular (LV) mass, and whether LV mass reduction is caused by atrophy. BACKGROUND The LV in patients with CTEPH is underfilled (unloaded). LV unloading may cause atrophic remodeling that is associated with diastolic and systolic dysfunction. METHODS We studied LV mass using cardiac magnetic resonance imaging (MRI) in 36 consecutive CTEPH patients (before/after pulmonary endarterectomy [PEA]) and 11 healthy volunteers selected to match age and sex of patients. We studied whether LV atrophy is present in monocrotaline (MCT)-injected rats with RVF or controls by measuring myocyte dimensions and performing in situ hybridization. RESULTS At baseline, CTEPH patients with RVF had significantly lower LV free wall mass indexes than patients without RVF (35 ± 6 g/m(2) vs. 44 ± 7 g/m(2), p = 0.007) or volunteers (42 ± 6 g/m(2), p = 0.006). After PEA, LV free wall mass index increased (from 38 ± 6 g/m(2) to 44 ± 9 g/m(2), p = 0.001), as right ventricular (RV) ejection fraction improved (from 31 ± 8% to 56 ± 12%, p < 0.001). Compared with controls, rats with RVF had reduced LV free wall mass and smaller LV free wall myocytes. Expression of atrial natriuretic peptide was higher, whereas that of α-myosin heavy chain and sarcoplasmic reticulum calcium ATPase-2 were lower in RVF than in controls, both in RV and LV. CONCLUSIONS RVF in patients with CTEPH is associated with reversible reduction in LV free wall mass. In a rat model of RVF, myocyte shrinkage due to atrophic remodeling contributed to reduction in LV free wall mass.

Genetically determined differences in sodium current characteristics modulate conduction disease severity in mice with cardiac sodium channelopathy.

Conduction slowing of the electric impulse that drives the heartbeat may evoke lethal cardiac arrhythmias. Mutations in SCN5A, which encodes the pore-forming cardiac sodium channel &agr; subunit, are associated with familial arrhythmia syndromes based on conduction slowing. However, disease severity among mutation carriers is highly variable. We hypothesized that genetic modifiers underlie the variability in conduction slowing and disease severity. With the aim of identifying such modifiers, we studied the Scn5a1798insD/+ mutation in 2 distinct mouse strains, FVB/N and 129P2. In 129P2 mice, the mutation resulted in more severe conduction slowing particularly in the right ventricle (RV) compared to FVB/N. Pan-genomic mRNA expression profiling in the 2 mouse strains uncovered a drastic reduction in mRNA encoding the sodium channel auxiliary subunit &bgr;4 (Scn4b) in 129P2 mice compared to FVB/N. This corresponded to low to undetectable &bgr;4 protein levels in 129P2 ventricular tissue, whereas abundant &bgr;4 protein was detected in FVB/N. Sodium current measurements in isolated myocytes from the 2 mouse strains indicated that sodium channel activation in myocytes from 129P2 mice occurred at more positive potentials compared to FVB/N. Using computer simulations, this difference in activation kinetics was predicted to explain the observed differences in conduction disease severity between the 2 strains. In conclusion, genetically determined differences in sodium current characteristics on the myocyte level modulate disease severity in cardiac sodium channelopathies. In particular, the sodium channel subunit &bgr;4 (SCN4B) may constitute a potential genetic modifier of conduction and cardiac sodium channel disease.

Late ventricular arrhythmias during acute regional ischemia in the isolated blood perfused pig heart Role of electrical cellular coupling

OBJECTIVE Acute ischemia comes with two phases of life-threatening arrhythmias, early (within 10 minutes, 1A) and late (after about 15 minutes, 1B). The mechanism of the latter is unknown and in this paper, we test the hypothesis that a phase of intermediate coupling between surviving epicardium and inexcitable midmyocardium underlies 1B arrhythmias. METHODS Pig hearts (n=26) were retrogradely perfused with a blood Tyrodes mixture. The left anterior descending artery was occluded. We investigated (1) inducibility of ventricular fibrillation (VF) with programmed stimulation, (2) tissue impedance (Rt) heterogeneity within the ischemic zone, (3) multiple subepicardial and midmyocardial electrograms, (4) subepicardial lactate dehydrogenase (LDH) and glycogen content. RESULTS In nine of ten hearts, one--three premature stimuli caused VF between 14 and 53 min of ischemia. This typically happened when the Rt of the ischemic zone had increased up to 40% of its final value. More uncoupling terminated the period of VF inducibility. The excitability of the surviving subepicardial layer was depressed during the same period with partial uncoupling, but recovered when the uncoupling from the midmyocardium had progressed further. CONCLUSIONS We show that 1B-VF can be induced within a distinct time window and coincides with a distinct range of Rt rise. Subepicardium is electrically depressed, presumably through coupling with midmyocardium, complete uncoupling causes subepicardial recovery and terminates the substrate for 1B-VF. Hence, we suggest that the substrate for 1B-VF consists of intermediate coupling of subepicardium and midmyocardium.