Mohamed Boutjdir

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.

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.

Intestine-specific MTP and global ACAT2 deficiency lowers acute cholesterol absorption with chylomicrons and HDLs.

Intestinal cholesterol absorption involves the chylomicron and HDL pathways and is dependent on microsomal triglyceride transfer protein (MTP) and ABCA1, respectively. Chylomicrons transport free and esterified cholesterol, whereas HDLs transport free cholesterol. ACAT2 esterifies cholesterol for secretion with chylomicrons. We hypothesized that free cholesterol accumulated during ACAT2 deficiency may be secreted with HDLs when chylomicron assembly is blocked. To test this, we studied cholesterol absorption in mice deficient in intestinal MTP, global ACAT2, and both intestinal MTP and global ACAT2. Intestinal MTP ablation significantly increased intestinal triglyceride and cholesterol levels and reduced their transport with chylomicrons. In contrast, global ACAT2 deficiency had no effect on triglyceride absorption but significantly reduced cholesterol absorption with chylomicrons and increased cellular free cholesterol. Their combined deficiency reduced cholesterol secretion with both chylomicrons and HDLs. Thus, contrary to our hypothesis, free cholesterol accumulated in the absence of MTP and ACAT2 is unavailable for secretion with HDLs. Global ACAT2 deficiency causes mild hypertriglyceridemia and reduces hepatosteatosis in mice fed high cholesterol diets by increasing hepatic lipoprotein production by unknown mechanisms. We show that this phenotype is preserved in the absence of intestinal MTP in global ACAT2-deficient mice fed a Western diet. Further, we observed increases in hepatic MTP activity in these mice. Thus, ACAT2 deficiency might increase MTP expression to avoid hepatosteatosis in cholesterol-fed animals. Therefore, ACAT2 inhibition might avert hepatosteatosis associated with high cholesterol diets by increasing hepatic MTP expression and lipoprotein production.

All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity

Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells significantly increased not only SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Moreover, we measured CPE synthase Km and Vmax for SMS1, SMS2, and SMSr using different NBD ceramides. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo.

Sodium overload due to a persistent current that attenuates the arrhythmogenic potential of a novel LQT3 mutation

Long QT syndrome (LQTS) is a congenital abnormality of cardiac repolarization that manifests as a prolonged QT interval on 12-lead electrocardiograms (ECGs). The syndrome may lead to syncope and sudden death from ventricular tachyarrhythmias known as torsades de pointes. An increased persistent Na+ current is known to cause a Ca2+ overload in case of ischemia for example. Such increased Na+ persistent current is also usually associated to the LQT3 syndrome. The purpose of this study was to investigate the pathological consequences of a novel mutation in a family affected by LQTS. The impact of biophysical defects on cellular homeostasis are also investigated. Genomic DNA was extracted from blood samples, and a combination of PCR and DNA sequencing of several LQTS-linked genes was used to identify mutations. The mutation was reproduced in vitro and was characterized using the patch clamp technique and in silico quantitative analysis. A novel mutation (Q1476R) was identified on the SCN5A gene encoding the cardiac Na+ channel. Cells expressing the Q1476R mutation exhibited biophysical alterations, including a shift of SS inactivation and a significant increase in the persistent Na+ current. The in silico analysis confirmed the arrhythmogenic character of the Q1476R mutation. It further revealed that the increase in persistent Na+ current causes a frequency-dependent Na+ overload in cardiomyocytes co-expressing WT and mutant Nav1.5 channels that, in turn, exerts a moderating effect on the lengthening of the action potential (AP) duration caused by the mutation. The Q1476R mutation in SCN5A results in a three-fold increase in the window current and a persistent inward Na+ current. These biophysical defects may expose the carrier of the mutation to arrhythmias that occur preferentially in the patient at rest or during tachycardia. However, the Na+ overload counterbalances the gain-of-function of the mutation and is beneficial in that it prevents severe arrhythmias at intermediate heart rates.

Calreticulin Negatively Regulates the Surface Expression of α1D L-Type Calcium Channel

Background: Quality control of several proteins is strictly regulated by molecular chaperones in the endoplasmic reticulum (ER). Calreticulin, an ER Ca2+ binding chaperone, has been shown to regulate the surface expression of several membrane proteins including the cyctic fibrosis transmembrane conductance regulator (CFTR) which under oxidative stress results in its internalization and proteasomal degredation. Decrease of L-type Ca current and channel protein has been described in autoimmune associated congenital heart block. Here, we demonstrated a novel mechanism of down-regulation of α1D L-type Ca channel using native human fetal cardiac cells and tsA201 cell line.Methods and Results: Using Confocal microscopy, we found surface staining of calreticulin on cultured human fetal cardiomyocytes (HFC) gestational age 18-24 weeks. Coimmunoprecipitation from HFC using anti-α1D antibody, and probing with anti-calreticulin antibody revealed a 46 kDa band corresponding to calreticulin. Overexpressing calreticulin in human embryonic kidney cells (tsA201) resulted in a decrease in surface expression of α1D L-type Ca Channel. Electrophysiological studies showed that co-transfection of calreticulin with α1D L-type Ca Channel led to 55% inhibition of the α1D Ca current expressed in tsA201 cells.Conclusions: These results show the first evidence that calreticulin: 1) is found on the cell surface of human fetal cardiomyocytes; 2) is coimmunoprecipitated with α1D L-type Ca Channel; 3) negatively regulates α1D surface expression; 4) decreases α1D Ca current in tsA201 cells co-expressed with α1D and calreticulin. The data demonstrated a novel mechanism of modulation of α1D Ca channel, which may be involved in numerous pathological settings such as congenital heart block.