Steven Poelzing

SCN5A Polymorphism Restores Trafficking of a Brugada Syndrome Mutation on a Separate Gene

Background— Brugada syndrome is associated with a high risk of sudden cardiac death and is caused by mutations in the cardiac voltage-gated sodium channel gene. Previously, the R282H-SCN5A mutation in the sodium channel gene was identified in patients with Brugada syndrome. In a family carrying the R282H-SCN5A mutation, an asymptomatic individual had a common H558R-SCN5A polymorphism and the mutation on separate chromosomes. Therefore, we hypothesized that the polymorphism could rescue the mutation. Methods and Results— In heterologous cells, expression of the mutation alone did not produce sodium current. However, coexpressing the mutation with the polymorphism produced significantly greater current than coexpressing the mutant with the wild-type gene, demonstrating that the polymorphism rescues the mutation. Using immunocytochemistry, we demonstrated that the R282H-SCN5A construct can traffic to the cell membrane only in the presence of the H558R-SCN5A polymorphism. Using fluorescence resonance energy transfer and protein fragments centered on H558R-SCN5A, we demonstrated that cardiac sodium channels preferentially interact when the polymorphism is expressed on one protein but not the other. Conclusions— This study suggests a mechanism whereby the Brugada syndrome has incomplete penetrance. More importantly, this study suggests that genetic polymorphisms may be a potential target for future therapies aimed at rescuing specific dysfunctional protein channels.

Interstitial volume modulates the conduction velocity-gap junction relationship

Cardiac conduction through gap junctions is an important determinant of arrhythmia susceptibility. Yet, the relationship between degrees of G(j) uncoupling and conduction velocity (θ) remains controversial. Conflicting results in similar experiments are normally attributed to experimental differences. We hypothesized that interstitial volume modulates conduction velocity and its dependence on G(j). Interstitial volume (V(IS)) was quantified histologically from guinea pig right ventricle. Optical mapping was used to quantify conduction velocity and anisotropy (AR(θ)). Albumin (4 g/l) decreased histologically assessed V(IS), increased transverse θ by 71 ± 10%, and lowered AR(θ). Furthermore, albumin did not change isolated cell size. Conversely, mannitol increased V(IS), decreased transverse θ by 24 ± 4%, and increased AR(θ). Mannitol also decreased cell width by 12%. Furthermore, mannitol was associated with spontaneous ventricular tachycardias in three of eight animals relative to zero of 15 during control. The θ-G(j) relationship was assessed using the G(j) uncoupler carbenoxolone (CBX). Whereas 13 μM CBX did not significantly affect θ during control, it slowed transverse θ by 38 ± 9% during mannitol (edema). These data suggest changes in V(IS) modulate θ, AR(θ), and the θ-G(j) relationship and thereby alter arrhythmia susceptibility. Therefore, V(IS) may underlie arrhythmia susceptibility, particularly in diseases associated with gap junction remodeling.

Mechanisms of Cardiac Conduction: A History of Revisions

Cardiac conduction is the process by which electrical excitation spreads through the heart, triggering individual myocytes to contract in synchrony. Defects in conduction disrupt synchronous activation and are associated with life-threatening arrhythmias in many pathologies. Therefore, it is scarcely surprising that this phenomenon continues to be the subject of active scientific inquiry. Here we provide a brief review of how the conceptual understanding of conduction has evolved over the last century and highlight recent, potentially paradigm-shifting developments.

The perinexus: Sign-post on the path to a new model of cardiac conduction?

The perinexus is a recently identified microdomain surrounding the cardiac gap junction that contains elevated levels of connexin43 and the sodium channel protein, Nav1.5. Ongoing work has established a role for the perinexus in regulating gap junction aggregation. However, recent studies have raised the possibility of a perinexal contribution at the gap junction cleft to intercellular propagation of action potential via non-electrotonic mechanisms. The latter possibility could modify the current theoretical understanding of cardiac conduction, help explain paradoxical experimental findings, and open up entirely new avenues for antiarrhythmic therapy. We review recent structural insights into the perinexus and its potential novel functional role in cardiac-excitation spread, highlighting presently unanswered questions, the evidence for ephaptic conduction in the heart and how structural insights may help complete this picture.

Status epilepticus induces cardiac myofilament damage and increased susceptibility to arrhythmias in rats.

Status epilepticus (SE) is a seizure or series of seizures that persist for >30 min and often results in mortality. Death rarely occurs during or immediately following seizure activity, but usually within 30 days. Although ventricular arrhythmias have been implicated in SE-related mortality, the effects of this prolonged seizure activity on the cardiac function and susceptibility to arrhythmias have not been directly investigated. We evaluated myocardial damage, alterations in cardiac electrical activity, and susceptibility to experimentally induced arrhythmias produced by SE in rats. SE resulted in seizure-related increases in blood pressure, heart rate, and the first derivative of pressure, as well as modest, diffuse myocyte damage assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining. Ten to twelve days following seizures, electrocardiographic recordings showed arrhythmogenic alterations in cardiac electrical activity, denoted by prolonged QT interval corrected for heart rate and QT dispersion. Finally, SE increased susceptibility to experimentally induced (intravenous aconitine) cardiac arrhythmias. These data suggest that SE produces tachycardic ischemia following the activation of the sympathetic nervous system, resulting in cardiac myofilament damage, arrhythmogenic alterations in cardiac electrical activity, and increased susceptibility to ventricular arrhythmias.

Extracellular sodium and potassium levels modulate cardiac conduction in mice heterozygous null for the Connexin43 gene

Several studies have disagreed on measurements of cardiac conduction velocity (CV) in mice with a heterozygous knockout of the connexin gene Gja1—a mutation that reduces the gap junction (GJ) protein, Connexin43 (Cx43), by 50 %. We noted that perfusate ionic composition varied between studies and hypothesized that extracellular ionic concentration modulates CV dependence on GJs. CV was measured by optically mapping wild-type (WT) and heterozygous null (HZ) hearts serially perfused with solutions previously associated with no change (Solution 1) or CV slowing (Solution 2). In WT hearts, CV was similar for Solutions 1 and 2. However, consistent with the hypothesis, Solution 2 in HZ hearts slowed transverse CV (CVT) relative to Solution 1. Previously, we showed CV slowing in a manner consistent with ephaptic conduction correlated with increased perinexal inter-membrane width (WP) at GJ edges. Thus, WP was measured following perfusion with systematically adjusted [Na+]o and [K+]o in Solutions 1 and 2. A wider WP was associated with reduced CVT in WT and HZ hearts, with the greatest effect in HZ hearts. Increasing [Na+]o increased CVT only in HZ hearts. Increasing [K+]o slowed CVT in both WT and HZ hearts with large WP but only in HZ hearts with narrow WP. Conclusion: When perinexi are wide, decreasing excitability by modulating [Na+]o and [K+]o increases CV sensitivity to reduced Cx43. By contrast, CV is less sensitive to Cx43 and ion composition when perinexi are narrow. These results are consistent with cardiac conduction dependence on both GJ and non-GJ (ephaptic) mechanisms.

A novel strategy using cardiac sodium channel polymorphic fragments to rescue trafficking-deficient SCN5A mutations.

Background— Brugada syndrome (BrS) is associated with mutations in the cardiac sodium channel (Nav1.5). We previously reported that the function of a trafficking-deficient BrS Nav1.5 mutation, R282H, could be restored by coexpression with the sodium channel polymorphism H558R. Here, we tested the hypothesis that peptide fragments from Nav1.5, spanning the H558R polymorphism, can be used to restore trafficking of trafficking-deficient BrS sodium channel mutations. Methods and Results— Whole-cell patch clamping revealed that cotransfection in human embryonic kidney (HEK293) cells of the R282H channel with either the 40- or 20-amino acid cDNA fragments of Nav1.5 containing the H558R polymorphism restored trafficking of this mutant channel. Fluorescence resonance energy transfer suggested that the trafficking-deficient R282H channel was misfolded, and this was corrected on coexpression with R558-containing peptides that restored trafficking of the R282H channel. Importantly, we also expressed the peptide spanning the H558R polymorphism with 8 additional BrS Nav1.5 mutations with reduced currents and demonstrated that the peptide was able to restore significant sodium currents in 4 of them. Conclusions— In the present study, we demonstrate that small peptides, spanning the H558R polymorphism, are sufficient to restore the trafficking defect of BrS-associated Nav1.5 mutations. Our findings suggest that it might be possible to use short cDNA constructs as a novel strategy tailored to specific disease-causing mutants of BrS.

Neuronal Na+ channel blockade suppresses arrhythmogenic diastolic Ca2+ release

AIMS Sudden death resulting from cardiac arrhythmias is the most common consequence of cardiac disease. Certain arrhythmias caused by abnormal 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+ and membrane potential imaging in ventricular myocytes 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; nNav) 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.

Gap junction heterogeneity as mechanism for electrophysiologically distinct properties across the ventricular wall

Gap junctions are critical to maintaining synchronized impulse propagation and repolarization. Heterogeneous expression of the principal ventricular gap junction protein connexin43 (Cx43) is associated with action potential duration (APD) dispersion across the anterior ventricular wall. Little is known about Cx43 expression patterns and their disparate impact on regional electrophysiology throughout the heart. We aimed to determine whether the anterior and posterior regions of the heart are electrophysiologically distinct. Multisegment, high-resolution optical mapping was performed in canine wedge preparations harvested separately from the anterior left ventricle (aLV; n = 8) and posterior left ventricle (pLV; n = 8). Transmural APD dispersion was significantly greater on the aLV than the pLV (45 +/- 13 vs. 26 +/- 8.0 ms; P < 0.05). Conduction velocity dispersion was also significantly higher (P < 0.05) across the aLV (39 +/- 7%) than the pLV (16 +/- 3%). Carbenoxolone perfusion significantly enhanced APD and conduction velocity dispersion on the aLV (by 1.53-fold and 1.36-fold, respectively), but not the pLV (by 1.27-fold and 1.2-fold, respectively), and produced a 4.2-fold increase in susceptibility to inducible arrhythmias in the aLV. Confocal immunofluorescence microscopy revealed significantly (P < 0.05) greater transmural dispersion of Cx43 expression on the aLV (44 +/- 10%) compared with the pLV wall (8.3 +/- 0.7%), suggesting that regional expression of Cx43 expression patterns may account for regional electrophysiological differences. Computer simulations affirmed that localized uncoupling at the epicardial-midmyocardial interface is sufficient to produce APD gradients observed on the aLV. These data demonstrate that the aLV and pLV differ importantly with respect to their electrophysiological properties and Cx43 expression patterns. Furthermore, local underexpression of Cx43 is closely associated with transmural electrophysiological heterogeneity on the aLV. Therefore, regional and transmural heterogeneous Cx43 expression patterns may be an important mechanism underlying arrhythmia susceptibility, particularly in disease states where gap junction expression is altered.

NCX is an important determinant for premature ventricular activity in a drug-induced model of Andersen–Tawil syndrome

AIMS Andersen-Tawil syndrome (ATS1)-associated ventricular arrhythmias are initiated by premature ventricular activity (PVA) resulting from diastolic Ca(2+) (Ca(D)) accumulation. We hypothesized that relatively high Na(+)-Ca(2+) exchanger (NCX) expression coupled with slower Ca(2+) uptake may constitute an arrhythmogenic substrate during drug-induced ATS1 (DI-ATS1). METHODS AND RESULTS DI-ATS1 was induced with 10 µmol/L BaCl(2) and 2 mmol/L [K(+)](o). Ca(2+) transients and action potentials were optically mapped from Langendorff-perfused guinea pig ventricles. Intracellular Ca(2+) handling was modulated by either direct NCX inhibition with 5 µmol/L KB-R7943 or by sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) inhibition with cyclopiazonic acid (CPA). During DI-ATS1, PVA was more frequent in left ventricular (LV)-base (LVB) vs. LV-apex (LVA) (2.2 ± 0.8 vs. 0.6 ± 0.3 PVA/10 min), consistent with greater Ca(D) (1.65 ± 0.13 vs. 1.42 ± 0.09 normalized-Ca(D) units) and western blot-assessed NCX protein expression (81.2 ± 30.9%) in LVB relative to LVA. Further, regions of high NCX (LVB) evidenced a shorter PVA coupling interval relative to regions of low NCX expression (LVA, 67.7 ± 3.5 vs. 78.5 ± 3.6%). Inhibiting NCX during DI-ATS1 lowered the incidence of ventricular tachycardias (VTs, 0 vs. 25%) and PVA (1.5 ± 0.4 vs. 4.3 ± 1.4 PVA/10 min), but it did not affect PVA coupling intervals in LVB nor LVA (70.8 ± 4.3 vs. 73.8 ± 2.5%). Conversely, inhibition of SERCA2a with CPA, thereby increasing the role of NCX in Ca(2+) handling, significantly increased the incidence of VTs and PVA relative to DI-ATS1 alone, while decreasing the PVA coupling interval in all regions. CONCLUSION PVA preferentially occurs in regions of enhanced NCX expression with relatively slower Ca(2+) uptake and during perfusion of CPA which further reduces sarcoplasmic reticular Ca(2+) uptake.