Yongxia Qu

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.

Gene expression of SERCA2a and L- and T-type Ca channels during human heart development.

In this study we report, for the first time, on the gene expression of human cardiac SERCA2a, L-type (α1C) and T-type (α1H) Ca channels during development, using RNase protection assay, relative quantitative RT-PCR and Western blot. Human hearts during early gestation (8- to 20-wk gestation), neonatal (1- to 4-d-old) and adult (18- to 48-year-old) stages were used. The results show that T-type Ca channel α1H subunit mRNA decreased and that L-type Ca channel α1C subunit mRNA increased with development. While the levels of sarcoplasmic reticulum ATPase (SERCA2a) mRNA did not significantly change with development, its protein levels increased with development. In conclusion, SERCA2a, L-type and T-type Ca channel transcripts were detected as early as 8-wk gestation. Defining the profile of Ca handling proteins during development is important to the understanding of excitation-contraction (EC)-coupling of the developing human heart.

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.

Gene expression of Na+/Ca2+ exchanger during development in human heart

OBJECTIVE In immature animal hearts, lower activity of sarcoplasmic reticulum and lower densities of Ca2+ channels highlight the potentially vital role of the Na+/Ca2+ exchanger (NCX) to excitation-contraction coupling. To date, studies on NCX expression have been restricted to late developmental stages. The distribution and gene expression of NCX during early ontogeny is not known, especially in humans. In the present report, we systematically characterized changes in NCX gene expression in human heart during development, with particular emphasis in early ontogeny. METHODS Human hearts during early gestation (9- to 20-week gestation), neonatal (1 to 2 days after birth) and adulthood (18-40 years old) were used. NCX mRNA levels were studied using RNase Protection Assay (RPA) and NCX protein levels were assessed by Western blot. Wet weight was also used as the tissue base. Immunolocalization studies using confocal microscopy were performed in isolated fetal cardiac myocytes. RESULTS Normalization of NCX mRNA derived from ventricles against an early gestational age (10-week gestation) shows that NCX mRNA levels nominally increased from 1 to 1.13 at 19-week gestation then decreased to 0.74 (P < 0.05) at neonate and further decreased to 0.23 (P < 0.05) at adult stages. NCX protein levels increased from 1 at 9-week gestation to 3 (P < 0.05) at 20-week gestation and then decreased to 1.8 (P < 0.05) at neonate and to 1.87 (P < 0.05) at adult stages. Confocal imaging of fetal cardiac myocytes revealed intense homogeneous membrane staining and abundance of NCX protein at this stage. CONCLUSIONS The data demonstrate changes in NCX transcript and NCX protein levels as well as total RNA and proteins during human heart development. Per wet weight, NCX mRNA was 4.5 times greater at early fetal than adult stages and NCX protein was 2 times greater at adult than the early fetal stage indicating considerable post-transcriptional regulation. These findings provide new insights into the understanding of temporal changes in NCX in the developing heart at the gene level. The functional significance remains to be determined.

Down-regulation of L-type calcium channel in pups born to 52 kDa SSA/Ro immunized rabbits

Congenital heart block is considered a model of passively acquired autoimmune disease in which the mother generates anti‐SSA/Ro and/or anti‐SSB/La antibodies that cross the placenta and presumably injure the heart of developing fetus. CHB is accompanied by ECG abnormalities including AV block, sinus bradycardia, and ventricular dysfunction. Our previous data indicate that these abnormalities are caused by maternal autoantibody‐mediated disturbance of L‐type Ca channels. To investigate the consequence of chronic exposure of L‐type Ca channels in newborn pups to maternal autoantibodies during pregnancy, we immunized female rabbits with human 52 kDa‐SSA/Ro (Ro52) recombinant protein. ECG revealed that pups from the immunized group had varying degrees of conduction defects. In addition, ICaL density and protein were reduced in hearts of pups from the immunized group. Sera and purified IgG from immunized rabbits inhibited IBa recorded from oocytes with expressed α1C and β2a subunits of L‐type Ca channel. Pups born to Ro52 immunized mothers exhibited down‐regulation of L‐type calcium channels in heart. The data provide new insight into the pathogenesis of congenital heart block.—Xiao, C.‐Q., Qu, Y., Hu, K., Boutjdir, M. Down‐regulation of L‐type calcium channel in pups born to 52 kDa SSA/Ro immunized rabbits. FASEB J. 15, 1539–1545 (2001)

Impaired Ca2+ homeostasis is associated with atrial fibrillation in the α1D L-type Ca2+ channel KO mouse

The novel alpha1D Ca2+ channel together with alpha1C Ca2+ channel contribute to the L-type Ca2+ current (I(Ca-L)) in the mouse supraventricular tissue. However, its functional role in the heart is just emerging. We used the alpha1D gene knockout (KO) mouse to investigate the electrophysiological features, the relative contribution of the alpha1D Ca2+ channel to the global I(Ca-L), the intracellular Ca2+ transient, the Ca2+ handling by the sarcoplasmic reticulum (SR), and the inducibility of atrial fibrillation (AF). In vivo and ex vivo ECG recordings from alpha1D KO mice demonstrated significant sinus bradycardia, atrioventricular block, and vulnerability to AF. The wild-type mice showed no ECG abnormalities and no AF. Patch-clamp recordings from isolated alpha1D KO atrial myocytes revealed a significant reduction of I(Ca-L) (24.5%; P < 0.05). However, there were no changes in other currents such as I(Na), I(Ca-T), I(K), I(f), and I(to) and no changes in alpha1C mRNA levels of alpha1D KO atria. Fura 2-loaded atrial myocytes showed reduced intracellular Ca2+ transient (approximately 40%; P < 0.05) and rapid caffeine application caused a 17% reduction of the SR Ca2+ content (P < 0.05) and a 28% reduction (P < 0.05) of fractional SR Ca2+ release in alpha1D KO atria. In conclusion, genetic deletion of alpha1D Ca2+ channel in mice results in atrial electrocardiographic abnormalities and AF vulnerability. The electrical abnormalities in the alpha1D KO mice were associated with a decrease in the total I(Ca-L) density, a reduction in intracellular Ca2+ transient, and impaired intracellular Ca2+ handling. These findings provide new insights into the mechanism leading to atrial electrical dysfunction in the alpha1D KO mice.

Rescue and Worsening of Congenital Heart Block-Associated Electrocardiographic Abnormalities in Two Transgenic Mice

Transgenic Murine Models of CHB. Introduction: Congenital heart block (CHB) is a passively acquired autoimmune disease considered to be due to the transfer of maternal autoantibodies, anti‐SSA/Ro –SSB/La, to the fetus resulting in atrioventricular (AV) block and sinus bradycardia. We previously established a murine model for CHB where pups born to immunized wild‐type (WT) mothers exhibited electrocardiographic abnormalities similar to those seen in CHB and demonstrated inhibition of L‐type Ca channels (LTCCs) by maternal antibodies. Here, we hypothesize that overexpression of LTCC should rescue, whereas knockout of LTCC should worsen the electrocardiographic abnormalities in mice.

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.

Phosphorylation of the Consensus Sites of Protein Kinase A on α1D L-type Calcium Channel

The novel α1D L-type Ca2+ channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased α1D Ca2+ channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the α1 subunit of the α1D Ca2+ channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the α1 subunit of α1D Ca2+ channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the α1 subunit of α1D Ca2+ channel. These novel findings provide new insights into the autonomic regulation of the α1D Ca2+ channel in the heart.