Yuankun Yue

Novel Molecular Mechanism Involving α1D (Cav1.3) L-Type Calcium Channel in Autoimmune-Associated Sinus Bradycardia

Background—Congenital heart block (CHB) is an autoimmune disease that affects fetuses/infants born to mothers with anti-Ro/La antibodies (positive IgG). Although the hallmark of CHB is complete atrioventricular block, sinus bradycardia has been reported recently in animal models of CHB. Interestingly, knockout of the neuroendocrine &agr;1D Ca channel in mice results in significant sinus bradycardia and atrioventricular block, a phenotype reminiscent to that seen in CHB. Here, we tested the hypothesis that the &agr;1D Ca channel is a novel target for positive IgG. Methods and Results—Reverse transcription–polymerase chain reaction, confocal indirect immunostaining, and Western blot data established the expression of the &agr;1D Ca channel in the human fetal heart. The effect of positive IgG on &agr;1D Ca current (ICa-L) was characterized in heterologous expression systems (tsA201 cells and Xenopus oocytes) because of the unavailability of &agr;1D-specific modulators. &agr;1D ICa-L activated at negative potentials (between −60 and −50 mV). Positive IgG inhibited &agr;1D ICa-L in both expression systems. This inhibition was rescued by a Ca channel activator, Bay K8644. No effect on &agr;1D ICa-L was observed with negative IgG and denatured positive IgG. Western blot data showed that positive IgG binds directly to &agr;1D Ca channel protein. Conclusions—The data are the first to demonstrate (1) expression of the &agr;1D Ca channel in human fetal heart, (2) inhibition of &agr;1D ICa-L by positive IgG, and (3) direct cross-reactivity of positive IgG with the &agr;1D Ca channel protein. Given that &agr;1D ICa-L activates at voltages within the pacemaker’s diastolic depolarization, inhibition of &agr;1D ICa-L in part may account for autoimmune-associated sinus bradycardia. In addition, Bay K8644 rescue of &agr;1D ICa-L inhibition opens new directions in the development of pharmacotherapeutic approaches in the management of CHB.

Functional Basis of Sinus Bradycardia in Congenital Heart Block

Abstract— Congenital heart block (CHB) is a conduction abnormality characterized by complete atrioventricular (AV) block. CHB affects fetuses and/or newborn of mothers with autoantibodies reactive with ribonucleoproteins 48-kDa SSB/La, 52-kDa SSA/Ro, and 60-kDa SSA/Ro. We recently established animal models of CHB and reported, for the first time, significant sinus bradycardia preceding AV block. This unexpected observation implies that the spectrum of conduction abnormalities extends beyond the AV node to also affect the SA node. To test this hypothesis, we investigated the functional basis of this sinus bradycardia by characterizing the effects of antibodies from mothers with CHB children (positive IgG) on ionic currents that are known to significantly contribute to spontaneous pacing in SA node cells. We recorded L- (ICa.L) and T- (ICa.T) type Ca2+, delayed rectifier K+ (IK), hyperpolarization-activated (If) currents, and action potentials (APs) from young rabbit SA node cells. We demonstrated that positive IgG significantly inhibited both ICa.T and ICa.L and induced sinus bradycardia but did not affect If and IK. Normal IgG from mothers with healthy children did not affect all the currents studied and APs. These results establish that IgG from mothers with CHB children causes substantial inhibition of ICa.T and ICa.L, two important pacemaker currents in rabbit SA node cells and point to both ICa.T and ICa.L as major players in the ionic mechanism by which maternal antibodies induce sinus bradycardia in CHB. These novel findings have important clinical significance and suggest that sinus bradycardia may be a potential marker in the detection and prevention of CHB. The full text of this article is available online at http://circres.ahajournals.org.

Congenital heart block: Identification of autoantibody binding site on the extracellular loop (domain I, S5–S6) of α1D L-type Ca channel

Congenital heart block (CHB) is an autoimmune disease associated with autoantibodies against intracellular ribonucleoproteins SSB/La and SSA/Ro. The hallmark of CHB is complete atrioventricular block. We have recently established that anti-SSA/Ro -SSB/La autoantibodies inhibit alpha(1D) L-type Ca current, I(Ca-L), and cross-react with the alpha(1D) Ca channel protein. This study aims at identifying the possible binding sites on alpha(1D) protein for autoantibodies from sera of mothers with CHB children. GST fusion proteins of the extracellular regions between the transmembrane segments (S5-S6) of each of the four alpha(1D) Ca channel protein domains I-IV were prepared and tested for reactivity with sera from mothers with CHB children and controls using ELISA. Sera containing anti-Ro/La autoantibodies from 118 mothers with CHB children and from 15 mothers with anti-Ro/La autoantibodies but have healthy children, and from 28 healthy mothers without anti-Ro/La autoantibodies and healthy children were evaluated. Seventeen of 118 (14.4%) sera from mothers with CHB children reacted with the extracellular loop of domain I S5-S6 region (E1). In contrast, only 2 of 28 (7%) of sera from healthy mothers (-anti-Ro/La) and healthy children reacted with E1 loop and none (0 of 15) of sera from healthy mothers (+anti-Ro/La) and healthy children reacted with the E1 loop. Preincubation of E1 loop with the positive sera decreased the O.D reading establishing the specificity of the response. Electrophysiological characterization of the ELISA positive sera and purified IgG showed inhibition (44.1% and 49.8%, respectively) of the alpha(1D) I(Ca-L) expressed in tsA201 cells. The inhibition was abolished when the sera were pre-incubated with E1 fusion protein. The results identified the extracellular loop of domain I S5-S6 of L-type Ca channel alpha(1D) subunit as a target for autoantibodies from a subset of mothers with CHB children. This novel finding provides insights into the potential development of therapeutic peptides that could bind to the pathogenic antibodies and prevent CHB.

Silencing of Cav1.2 gene in neonatal cardiomyocytes by lentiviral delivered shRNA.

Cav1.2 (alpha1C) and Cav1.3 (alpha1D) L-type Ca channels are co-expressed in the heart. To date, there are no pharmacological or biophysical tools to separate alpha1D from alpha1C Ca currents (I(Ca-L)) in cardiomyocytes. Here, we established a physiological model to study alpha1D I(Ca-L) in native myocytes using RNA interference. Transfection of rat neonatal cardiomyocytes (RNC) with alpha1C specific siRNA resulted in low silencing efficiency (50-60%) at the mRNA and protein levels. The use of lentivirus shRNA resulted in 100% transfection efficiency and 92% silencing of the alpha1C gene by real-time PCR and Western blot. Electrophysiological experiments showed that the total I(Ca-L) was similarly reduced by 80% in lentivirus transfected cells. Both biochemical and functional data demonstrated high transfection and silencing efficiency in the cardiomyocytes using lentiviral shRNA. This novel approach allows for the assessments of the roles of alpha1C and alpha1D Ca channels in native myocytes and could be used to examine their roles in physiological and pathological settings.

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

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.