Induction of autoimmune response to the extracellular loop of the HERG channel pore induces QTc prolongation in guinea-pigs.

KEY POINTS: Channelopathies of autoimmune origin are novel and are associated with corrected QT (QTc) prolongation and complex ventricular arrhythmias. We have recently demonstrated that anti-SSA/Ro antibodies from patients with autoimmune diseases and with QTc prolongation on the ECG target the human ether-à-go-go-related gene (HERG) K+ channel by inhibiting the corresponding current, IKr , at the pore region. Immunization of guinea-pigs with a peptide (E-pore peptide) corresponding to the extracellular loop region connecting the S5 and S6 segments of the HERG channel induces high titres of antibodies that inhibit IKr , lengthen the action potential and cause QTc prolongation on the surface ECG. In addition, anti-SSA/Ro-positive sera from patients with connective tissue diseases showed high reactivity to the E-pore peptide. The translational impact is the development of a peptide-based approach for the diagnosis and treatment of autoimmune-associated long QT syndrome. ABSTRACT: We recently demonstrated that anti-SSA/52 kDa Ro antibodies (Abs) from patients with autoimmune diseases and corrected QT (QTc) prolongation directly target and inhibit the human ether-à-go-go-related gene (HERG) K+ channel at the extracellular pore (E-pore) region, where homology with SSA/52 kDa Ro antigen was demonstrated. We tested the hypothesis that immunization of guinea-pigs with a peptide corresponding to the E-pore region (E-pore peptide) will generate pathogenic inhibitory Abs and cause QTc prolongation. Guinea-pigs were immunized with a 31-amino-acid peptide corresponding to the E-pore region of HERG. On days 10-62 after immunization, ECGs were recorded and blood was sampled for the detection of E-pore peptide Abs. Serum samples from patients with autoimmune diseases were evaluated for reactivity to E-pore peptide by enzyme-linked immunosorbent assay (ELISA), and histology was performed on hearts using Masson's Trichrome. Inhibition of the HERG channel was assessed by electrophysiology and by computational modelling of the human ventricular action potential. The ELISA results revealed the presence of high titres of E-pore peptide Abs and significant QTc prolongation after immunization. High reactivity to E-pore peptide was found using anti-SSA/Ro Ab-positive sera from patients with QTc prolongation. Histological data showed no evidence of fibrosis in immunized hearts. Simulations of simultaneous inhibition of repolarizing currents by anti-SSA/Ro Ab-positive sera showed the predominance of the HERG channel in controlling action potential duration and the QT interval. These results are the first to demonstrate that inhibitory Abs to the HERG E-pore region induce QTc prolongation in immunized guinea-pigs by targeting the HERG channel independently from fibrosis. The reactivity of anti-SSA/Ro Ab-positive sera from patients with connective tissue diseases with the E-pore peptide opens novel pharmacotherapeutic avenues in the diagnosis and management of autoimmune-associated QTc prolongation.

Arrhythmogenicity of Anti-Ro/SSA Antibodies in Patients With Torsades de Pointes.

BACKGROUND: In patients with autoimmune disease, anti-Ro/SSA antibodies (anti-Ro/SSA) are responsible for a novel autoimmune-associated long-QT syndrome by targeting the hERG potassium channel and inhibiting the related current (IKr). Because anti-Ro/SSA are also present in a significant proportion of healthy subjects and may be associated with torsades de pointes (TdP) arrhythmia, we tested the hypothesis that anti-Ro/SSA may represent a silent risk factor in patients developing TdP. METHODS AND RESULTS: Twenty-five consecutive patients who experienced TdP were prospectively collected independent of ongoing therapies and concomitant diseases. Anti-Ro/SSA were detected by fluoroenzyme immunoassay, immuno-Western blotting, and line-blot immunoassay. Purified IgGs from anti-Ro/SSA-positive and anti-Ro/SSA-negative patients were tested on IKr using HEK293 cells stably expressing the hERG channel. As expected, in TdP patients, many known corrected QT interval-prolonging risk factors were simultaneously present, including hypokalemia that was the most common (52%). Anti-Ro/SSA were present in 60% of the subjects, mostly the anti-Ro/SSA-52-kD subtype detected by immuno-Western blotting only. A history of autoimmune disease was found in only 2 of anti-Ro/SSA-positive patients. Experimental data demonstrated that purified anti-Ro/SSA-positive IgGs significantly inhibited IKr and cross reacted with hERG-channel proteins. Moreover, anti-Ro/SSA-positive sera exhibited high reactivity with a peptide corresponding to the hERG-channel pore-forming region. CONCLUSIONS: Anti-Ro/SSA may represent a clinically silent novel risk factor for TdP development via an autoimmune-mediated electrophysiological interference with the hERG channel. We propose that TdP patients may benefit from specific anti-Ro/SSA testing even in the absence of autoimmune diseases as immunomodulating therapies may be effective in shortening corrected QT interval and reducing TdP recurrence risk.

Pathogenesis of the Novel Autoimmune-Associated Long-QT Syndrome.

BACKGROUND: Emerging clinical evidence demonstrates high prevalence of QTc prolongation and complex ventricular arrhythmias in patients with anti-Ro antibody (anti-Ro Ab)-positive autoimmune diseases. We tested the hypothesis that anti-Ro Abs target the HERG (human ether-a-go-go-related gene) K(+) channel, which conducts the rapidly activating delayed K(+) current, IKr, thereby causing delayed repolarization seen as QT interval prolongation on the ECG. METHODS AND RESULTS: Anti-Ro Ab-positive sera, purified IgG, and affinity-purified anti-52kDa Ro Abs from patients with autoimmune diseases and QTc prolongation were tested on IKr using HEK293 cells expressing HERG channel and native cardiac myocytes. Electrophysiological and biochemical data demonstrate that anti-Ro Abs inhibit IKr to prolong action potential duration by directly binding to the HERG channel protein. The 52-kDa Ro antigen-immunized guinea pigs showed QTc prolongation on ECG after developing high titers of anti-Ro Abs, which inhibited native IKr and cross-reacted with guinea pig ERG channel. CONCLUSIONS: The data establish that anti-Ro Abs from patients with autoimmune diseases inhibit IKr by cross-reacting with the HERG channel likely at the pore region where homology between anti-52-kDa Ro antigen and HERG channel is present. The animal model of autoimmune-associated QTc prolongation is the first to provide strong evidence for a pathogenic role of anti-Ro Abs in the development of QTc prolongation. It is proposed that adult patients with anti-Ro Abs may benefit from routine ECG screening and that those with QTc prolongation should receive counseling about drugs that may increase the risk for life-threatening arrhythmias.

Early voltage/calcium uncoupling predestinates the duration of ventricular tachyarrhythmias during ischemia/reperfusion.

BACKGROUND: Abnormal intracellular calcium (Ca(i)) kinetics during ischemia/reperfusion (I/R) can alter membrane voltage (V(m)) and destabilize wavefront propagation. OBJECTIVE: We used optical mapping to investigate the hypothesis that early V(m)/Ca(i) uncoupling during a ventricular tachyarrhythmia (VT) can play a primary role in perpetuation of VT episodes. METHODS: Seventeen Langendorff-perfused guinea pig hearts were subjected to 15 min I/15 min R. Simultaneous optical recordings of V(m) and Ca(i) signals were obtained using a dual-photodiode array. Spatiotemporal entropy (E) was used to quantify differences in V(m)/Ca(i) kinetics during VT and compare wavefront topology during the first 500 ms of a VT episode. RESULTS: A total of 39 episodes of VT were analyzed; VT was classified as self-terminating (ST, n = 28) and non-self-terminating (NST, n = 11). The ST/VTs were further classified into short ST/VT (1 to 5 s in duration; n = 16) and long ST/VT (>5 s, n = 12). E values for NST/VTs were significantly higher than E values for both short and long ST/VTs separately as well as E values for ST/VTs as a group. Further, E values for long ST/VTs were significantly higher than E values for short ST/VTs. Wave breaks were consistently identified during periods of high E. CONCLUSION: High E during the first 500 ms of the onset of VT (the first 2 to 3 beats) is significantly correlated with long ST or NST episodes. This may be related to destabilization of wave propagation that helps to perpetuate VT. Early V(m)/Ca(i) uncoupling can predestinate the development of a malignant NST/VT.

Protective role of protein kinase C epsilon activation in ischemia-reperfusion arrhythmia.

PURPOSE: Ischemic heart disease carries an increased risk of malignant ventricular tachycardia (VT), fibrillation (VF), and sudden cardiac death. Protein kinase C (PKC) epsilon activation has been shown to improve the hemodynamics in hearts subjected to ischemia/reperfusion. However, very little is known about the role of epsilon PKC in reperfusion arrhythmias. Here we show that epsilon PKC activation is anti-arrhythmic and its inhibition is pro-arrhythmic. METHOD: Langendorff-perfused isolated hearts from epsilonPKC agonist (epsilonPKC activation), antagonist (epsilonPKC inhibition) transgenic (TG), and wild-type control mice were subjected to 30 min stabilization period, 10 min global ischemia, and 30 min reperfusion. Action potentials (APs) and calcium transients (CaiT) were recorded simultaneously at 37 degrees C using optical mapping techniques. The incidence of VT and VF was assessed during reperfusion. RESULTS: No VT/VF was seen in any group during the stabilization period in which hearts were perfused with Tyrode's solution. Upon reperfusion, 3 out of the 16 (19%) wild-type mice developed VT but no VF. In epsilonPKC antagonist group, in which epsilonPKC activity was downregulated, 10 out of 13 (76.9%) TG mice developed VT, of which six (46.2%) degenerated into sustained VF upon reperfusion. Interestingly, in epsilonPKC agonist mice, in which the activity of epsilonPKC was upregulated, no VF was observed and only 1 out of 12 mice showed only transient VT during reperfusion. During ischemia and reperfusion, CaiT decay was exceedingly slower in the antagonist mice compared to the other two groups. CONCLUSION: Moderate in vivo activation of epsilonPKC exerts beneficial antiarrhythmic effect vis-a-vis the lethal reperfusion arrhythmias. Abnormal CaiT decay may, in part, contribute to the high incidence of reperfusion arrhythmias in the antagonist mice. These findings have important implications for the development of PKC isozyme targeted therapeutics and subsequently for the treatment of ischemic heart diseases.

Localization and modulation of {alpha}1D (Cav1.3) L-type Ca channel by protein kinase A.

Alpha1D L-type Ca channel was assumed to be of neuroendocrine origin only; however, alpha1D L-type Ca channel knockout mice exhibit sinus bradycardia and atrioventricular block, indicating a distinct role of alpha1D in the heart. The presence and distribution of alpha1D Ca channel in the heart and its regulation by protein kinase A (PKA) are just emerging. Our objective was to examine the localization of alpha1D L-type Ca channel in rabbit and rat hearts and its modulation by PKA. Here, we show the exclusive presence of alpha1D Ca channel transcript in the sinoatrial node, atrioventricular node, and atria but not in the ventricle by RT-PCR and the expression of alpha1D Ca channel protein in atrial myocytes' sarcolemma by indirect immunostaining and Western blot. There is no significant difference in the expression level of alpha1D Ca channel in the left versus right atrium. Superfusion of membrane-permeable 8-bromo-cAMP resulted in a significant increase of the peak current density of alpha1D Ca current expressed in tsA201 cells. This increase was inhibited by the PKA inhibitor (PKI). Application of 8-bromo-cAMP also readily phosphorylated the alpha1D Ca channel protein. The results are first to demonstrate that PKA phosphorylation of L-type Ca channel alpha1D-subunit resulted in an increase of the alpha1D Ca channel activity. Together with the observation that alpha1D Ca channel is exclusively present in the sinoatrial node and atria, the findings suggest that alpha1D Ca channel plays a unique role in the sinoatrial tissue and is a target for sympathetic control of heart rhythm.

Experimental study of adenovirus vector mediated-hVEGF165 gene on prevention of restenosis after angioplasty.

This study evaluated the effects of adenovirus vector mediated human vascular endothelial growth factor-165 (hVEGF165) gene on prevention of restenosis after angioplasty. Rabbit models of bilateral carotid artery injury were established by balloon denudation. The recombinant adenoviruses containing hVEGF165 cDNA was directly injected into left side of the injured carotid arteries. On day 3 and week 3 after transfection the expression of VEGF was observed by RT-PCR and immunohistochemistry. The thrombokinesis, reendothelialization (rET) and intimal hyperplasia in carotid arteries were evaluated by computerized image analysis system 3 weeks after gene transfer. The changes in the VEGF gene-treated side were compared with the control side. Our results showed that 3 days and 3 weeks after hVEGF165 gene transfer the VEGF mRNA and antigen expression were detected in vivo. 3 weeks after the transfer, the carotid artery rET was markedly better in the VEGF gene-treated group compared with the control. The thrombokinesis, intima area/media area (I/M), maximal intimal and medial thicknesses (ITmax and MTmax) demonstrated a statistically significant decrease in arteries treated with VEGF gene as compared with the control group. It is concluded that VEGF gene transfer could be achieved by intra-arterial injection of recombinant adenoviruses. It might accelerate the restoration of endothelial integrity, inhibit thrombokinesis and attenuate intimal hyperplasia in the injured arteries after VEGF gene transfer. This procedure could be useful in preventing restenosis after angioplasty.