Mohamed Boutjdir, Ph.D.

Rescue and Worsening of Congenital Heart Block-Associated Electrocardiographic Abnormalities in Two Transgenic Mice
Karnabi E; Qu Y; Mancarella S; Boutjdir M
2011 Aug;22(8):922-30 L, Journal of cardiovascular electrophysiology
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. Methods and Results: Transgenic (TG) mice were immunized with SSA/Ro and SSB/La antigens. Pups born to immunized WT mothers had significantly greater sinus bradycardia and AV block compared to pups from nonimmunized WT. TG pups overexpressing LTCC had significantly less sinus bradycardia and AV block compared to their non-TG littermates and to pups born to immunized WT mothers. All LTCC knockout pups born to immunized mothers had sinus bradycardia, advanced degree of AV block, and decreased fetal parity. No sinus bradycardia or AV block were manifested in pups from control nonimmunized WT mothers. IgG from mothers with CHB children, but not normal IgG, completely inhibited intracellular Ca transient ([Ca](i) T) amplitude. Conclusions: Cardiac-specific overexpression of LTCC significantly reduced the incidence of AV block and sinus bradycardia in pups exposed to anti-SSA/Ro -SSB/La autoantibodies, whereas exposure of LTCC knockout pups to these autoantibodies significantly worsened the electrocardiographic abnormalities. These findings support the hypothesis that maternal antibodies inhibit LTCC and [Ca](i) T thus contributing to the development of CHB. Altogether, the results are relevant to the development of novel therapies for CHB. (J Cardiovasc Electrophysiol, Vol. pp. 1-9)
— id: 126653, year: 2011, vol: 22, page: 922, stat: Journal Article,

TRPC channels, an overarching Ca2+ paradigm in the developing heart
Qu Y.; Boutjdir M.
2011 ;92(2):189-190, Cardiovascular research
— id: 139759, year: 2011, vol: 92, page: 189, stat: Journal Article,

Perinatal and Postnatal Expression of Cav1.3 alpha1D Ca2+ Channel in the Rat Heart
Qu Y; Karnabi E; Ramadan O; Yue Y; Chahine M; Boutjdir M
2011 Jun;69(6):479-484, Pediatric research
The novel Cav1.3 (alpha1D) L-type Ca channel plays a significant role in sino-atrial, atrioventricular nodes function and in atrial fibrillation. However, the characterization of alpha1D Ca channel during heart development is very limited. We used real-time RT-PCR, Western blotting and indirect immunostaining to characterize the developmental expression and localization of alpha1D Ca channel in rat hearts. Both protein and mRNA levels of alpha1D Ca channel decreased postnatally. Two forms of alpha1D Ca channel protein (250 kD and 190 kD) were observed, with the full length (250kD) channel protein being predominant in the prenatal stages. Both Western blots and confocal imaging demonstrated that alpha1D Ca channel protein was expressed in both atria and ventricles at fetal and neonatal stages but was absent in the adult ventricles. Interestingly, alpha1D Ca channel was also found at the nucleus/perinucleus of immature, but not adult atrial cells. Furthermore, the nuclear staining was reproduced in adult atrial cell line, HL-1 cells, which possess immature properties. The data are first to show that alpha1D Ca channel has unique age-dependent expression profile and subcellular localization in the heart, suggesting a developmental stage dependent specific function. ABBREVIATIONS:
— id: 126652, year: 2011, vol: 69, page: 479, stat: Journal Article,

Role of calcium channels in congenital heart block
Karnabi, E; Boutjdir, M
2010 Sep;72(3):226-234, Scandinavian journal of immunology
Congenital heart block (CHB) is a conduction abnormality that affects hearts of foetuses and/or newborn to mothers with autoantibodies reactive with the intracellular soluble ribonucleoproteins 48-kD La, 52-kD Ro and 60-kD Ro. CHB carries substantial mortality and morbidity, with more than 60% of affected children requiring lifelong pacemakers. Several hypotheses have been proposed to explain the pathogenesis of CHB. These can be grouped under three main hypotheses: Apoptosis, Serotoninergic and Ca channel hypothesis. Here, we discuss these hypotheses and provide recent scientific thinking that will most likely dominate the future of this field of research
— id: 111594, year: 2010, vol: 72, page: 226, stat: Journal Article,

Congenital heart block: identification of autoantibody binding site on the extracellular loop (domain I, S5-S6) of alpha(1D) L-type Ca channel
Karnabi, Eddy; Qu, Yongxia; Wadgaonkar, Raj; Mancarella, Salvatore; Yue, Yuankun; Chahine, Mohamed; Clancy, Robert M; Buyon, Jill P; Boutjdir, Mohamed
2010 Mar;34(2):80-86, Journal of autoimmunity
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
— id: 114633, year: 2010, vol: 34, page: 80, stat: Journal Article,

Silencing of Cav1.2 gene in neonatal cardiomyocytes by lentiviral delivered shRNA
Karnabi, Eddy; Qu, Yongxia; Mancarella, Salvatore; Yue, Yuankun; Wadgaonkar, Raj; Boutjdir, Mohamed
2009 Jul 10;384(4):409-414, Biochemical & biophysical research communications
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
— id: 99214, year: 2009, vol: 384, page: 409, stat: Journal Article,

PKA Activation Regulates L-type Cav1.3 Calcium Channel in vivo
Ramadan, O; Boutjdir, M
2009 NOV 3 ;120(18):S626-S626, Circulation
— id: 106975, year: 2009, vol: 120, page: S626, stat: Journal Article,

Phosphorylation of the consensus sites of protein kinase A on alpha1D L-type calcium channel
Ramadan, Omar; Qu, Yongxia; Wadgaonkar, Raj; Baroudi, Ghayath; Karnabi, Eddy; Chahine, Mohamed; Boutjdir, Mohamed
2009 Feb 20;284(8):5042-5049, Journal of biological chemistry
The novel alpha(1D) L-type Ca(2+) 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 alpha(1D) Ca(2+) channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the alpha(1) subunit of the alpha(1D) Ca(2+) channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the alpha(1) subunit of alpha(1D) Ca(2+) 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 alpha(1) subunit of alpha(1D) Ca(2+) channel. These novel findings provide new insights into the autonomic regulation of the alpha(1D) Ca(2+) channel in the heart
— id: 126654, year: 2009, vol: 284, page: 5042, stat: Journal Article,

Role of subendocardial Purkinje network in triggering torsade de pointes arrhythmia in experimental long QT syndrome
Ben Caref, E; Boutjdir, Mohamed; Himel, Herman D; El-Sherif, Nabil
2008 Oct;10(10):1218-1223, Europace
AIMS: The present study addresses the controversy regarding the 'primary' role of the subendocardial Purkinje network in triggering torsade de pointes (TdP) ventricular tachyarrhythmia (VAs) in the long QT syndrome (LQTS). METHODS AND RESULTS: We investigated the well-established canine anthopleurin-A (AP-A) surrogate model of LQT3 to study the role of the subendocardial Purkinje network in triggering VAs. Three-dimensional activation and repolarization patterns were analysed from unipolar extracellular electrograms utilizing 64 plunge needle electrodes. In 6 dogs, the animals were placed on cardiopulmonary bypass and chemical ablation of the endocardial Purkinje network was obtained using Lugol's solution. Spontaneous VAs consistently developed in response to AP-A infusion and were triggered by a subendocardial focal activity acting on a substrate of spatial three-dimensional dispersion of repolarization. Endocardial ablation was considered successful by the development of complete atrioventricular block in the absence of ventricular escape rhythm. Following endocardial ablation spontaneous VAs were no longer observed. However, an appropriately coupled premature stimulus consistently induced re-entrant VAs. CONCLUSION: The present study strongly suggests that in the LQTS, focal activity generated in subendocardial Purkinje tissue is the primary, if not the only, trigger for TdP VAs by acting on a substrate of three-dimensional dispersion of myocardial repolarization to induce re-entrant excitation
— id: 126656, year: 2008, vol: 10, page: 1218, stat: Journal Article,

Protein kinase C activation inhibits alpha1D L-type Ca channel: a single-channel analysis
Chahine, M; Qu, Y; Mancarella, S; Boutjdir, M
2008 Feb;455(5):913-919, Pflugers archiv = European journal of physiology
The recently reported alpha1D Ca channel in the heart is known to be regulated by protein kinase C (PKC) at the whole cell level and has been implicated in atrial fibrillation. The biophysical basis of this regulation at the single-channel level is not known. Therefore, the effect of PKC activation was studied on alpha1D Ca channel expressed in tsA201 cells using cell-attached configuration. Unitary currents were recorded in the presence of 70 mM Ba2+ as the charge carrier at room temperature. Under basal condition, channel activity was rare and infrequent; however, Bay K 8644 (1 microM) induced channel openings with a conductance of 22.3 pS. Single channel analysis of open and closed time distributions were best fitted with a single exponential. PKC activation by 4alpha-phorbol 12-myristate 13-acetate (PMA; 10 nM), a phorbol ester derivative, resulted in a decrease in open probability and increase in closed-time without any significant effect on the conductance of the alpha1D Ca channel. This is consistent with a decreased entry of alpha1D Ca channel into open states in the presence of PMA. PMA effects could not be reproduced by 4-alpha Phorbol, an inactive PMA analogue. These data show, for the first time, (1) the alpha1D Ca channel activity at the single-channel level and (2) the biophysical basis by which PKC activation inhibits the alpha1D Ca channel. The shortening of the open-time and the lengthening of the closed-time constants and the increase in blank sweeps may explain the inhibition of the previously reported whole-cell alpha1D Ca current. Altogether, these data are essential for understanding the complex role of alpha1D Ca channel not only in physiological settings but also in pathological settings such as atrial fibrillation
— id: 135309, year: 2008, vol: 455, page: 913, stat: Journal Article,

Impaired Ca2+ homeostasis is associated with atrial fibrillation in the alpha1D L-type Ca2+ channel KO mouse
Mancarella, Salvatore; Yue, Yuankun; Karnabi, Eddy; Qu, Yongxia; El-Sherif, Nabil; Boutjdir, Mohamed
2008 Nov;295(5):H2017-H2024, American journal of physiology. Heart & circulatory physiology
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
— id: 126655, year: 2008, vol: 295, page: H2017, stat: Journal Article,

Protective role of intracellular zinc in myocardial ischemia/reperfusion is associated with preservation of protein kinase C isoforms
Karagulova, Gulnura; Yue, Yuankun; Moreyra, Abel; Boutjdir, Mohamed; Korichneva, Irina
2007 May;321(2):517-525, Journal of pharmacology & experimental therapeutics
The recent discovery of zinc signals and their essential role in the redox signaling network implies that zinc homeostasis and the function of zinc-containing proteins are probably altered as a result of oxidative stress, suggesting new targets for pharmacological intervention. We hypothesized that the level of intracellular labile zinc is changed in hearts subjected to ischemia/reperfusion (I/R) and investigated whether the maintenance of myocardial zinc status protected heart functions. Using fluorescent imaging, we demonstrated decreased levels of labile zinc in the I/R hearts. Phorbol 12-myristate 13-acetate, a known trigger of zinc release, liberated zinc ions in control hearts but failed to produce any increase in zinc levels in the I/R rat hearts. Adding the zinc ionophore pyrithione at reperfusion improved myocardial recovery up to 100% and reduced the incidence of arrhythmias more than 2-fold. This effect was dose-dependent, and high concentrations of zinc were toxic. Adding membrane-impermeable zinc chloride was ineffective. Hearts from rats receiving zinc pyrithione supplements in their diet fully recovered from I/R. The recovery was associated with the prevention of degradation of the two protein kinase C isoforms, delta and epsilon, during I/R. In conclusion, our results suggest a protective role of intracellular zinc in myocardial recovery from oxidative stress imposed by I/R. The data support the potential clinical use of zinc ionophores in the settings of acute redox stress in the heart.
— id: 72809, year: 2007, vol: 321, page: 517, stat: Journal Article,

Reduction of L-type Ca2+ current results in electrocardiographic abnormalities in alpha(1D) L-type Ca2+ channel knock out mice
Mancarella, S; Yue, Y; Ou, Y; Boutjdir, M
2007 OCT 16 ;116(16):276-276, Circulation
— id: 75967, year: 2007, vol: 116, page: 276, stat: Journal Article,

The dual-specificity kinases, TOPK and DYRK1A, are critical for oocyte maturation induced by wild-type--but not by oncogenic--ras-p21 protein
Qu, Yongxia; Adler, Victor; Izotova, Lara; Pestka, Sidney; Bowne, Wilbur; Michl, Josef; Boutjdir, Mohamed; Friedman, Fred K; Pincus, Matthew R
2007 ;12:5089-5097, Frontiers in biosciences
We have previously found that oncogenic ras-p21 and insulin, which activates wild-type ras-21 protein, both induce Xenopus laevis oocyte maturation that is dependent on activation of raf. However, oncogenic ras-p21 utilizes raf-dependent activation of the two classic raf targets, MEK and MAP kinase (MAPK or ERK) while insulin-activated wild-type ras-p21 does not depend on activation of these two kinases. Utilizing a microarray containing the entire Xenopus genome, we discovered two dual specificity kinases, T-Cell Origin Protein Kinase (TOPK), known to bind to raf and the nuclear kinase, DYRK1A, that are expressed at much higher levels in insulin-matured oocytes. Using SiRNA's directed against expression of both of these proteins, we now show that each inhibits insulin-but not oncogenic ras-p21-induced oocyte maturation. Control siRNA's have no effect on either agent in induction of maturation. We find that each SiRNA 'knocks down' expression of its target protein while not affecting expression of the other protein. These results suggest that both proteins are required for maturation induced by wild-type, but not oncogenic, ras-p21. They also suggest that oncogenic and wild-type ras-p21 utilize pathways that become divergent downstream of raf. On the basis of these findings, we propose a model for two signal transduction pathways by oncogenic and activated wild-type ras-p21 showing points of overlap and divergence
— id: 126657, year: 2007, vol: 12, page: 5089, stat: Journal Article,

RNase protection assay for quantifying gene expression levels
Qu, Yongxia; Boutjdir, Mohamed
2007 ;366:145-158, Methods in molecular biology
Quantifying the level of mRNA is central to the study of mammalian gene expression. Conventional approaches such as Northern blotting are often prone to low sensitivity and reproducibility. The RNase protection assay (RPA) provides a sensitive alternative for the detection and quantification of mRNA. The RPA is based on the hybridization in solution of a labeled single-stranded antisense RNA probe with a target mRNA. After hybridization, single-strand specific RNases are then used to digest away unhybridized RNA. The hybrid can be resolved by a denaturing gel. Subsequent detection will reveal the appropriate-sized gel band corresponding to the target mRNA. The major advantage of RPA is the high sensitivity and the simultaneous detection and quantification of multiple mRNA targets in a single RNA sample. The primary limitation of RPA is the lack of information on transcript size
— id: 126658, year: 2007, vol: 366, page: 145, stat: Journal Article,

Expression of skeletal muscle Na(V)1.4 Na channel isoform in canine cardiac Purkinje myocytes
Qu, Yongxia; Karnabi, Eddy; Chahine, Mohamed; Vassalle, Mario; Boutjdir, Mohamed
2007 Mar 30;355(1):28-33, Biochemical & biophysical research communications
BACKGROUND AND AIM: The action potential plateau of Purkinje fibers is particularly sensitive to tetrodotoxin (TTX) and this could be due to a TXX-sensitive Na(+) current. The expression of TTX-sensitive neuronal Na(V)1.1 and Na(V)1.2 isoforms has been reported in canine Purkinje myocytes. Our aim was to investigate by means of biochemical and functional techniques whether the TTX-sensitive skeletal Na(V)1.4 isoform is also expressed in canine cardiac Purkinje myocytes. METHODS AND RESULTS: Using Na(V)1.4 specific primers, a PCR product corresponding to Na(V)1.4 was amplified from canine Purkinje fibers RNA and confirmed by sequencing and megablast of the gene bank. Confocal indirect immunostaining using anti-Na(V)1.4 antibody demonstrates distinct sarcolemmal staining pattern compared to that of the cardiac isoform Na(V)1.5. Expression of Na(V)1.4 in tsA201 cells yielded a TTX-sensitive Na(+) current with an IC(50) of 10nM. CONCLUSIONS: These results demonstrate the expression of the TTX-sensitive Na(V)1.4 channel in canine cardiac Purkinje myocytes. This novel finding suggests a role of Na(V)1.4 channel in Purkinje myocytes and thus has important clinical implications for the mechanisms and management of ventricular arrhythmias originating in the Purkinje network
— id: 126659, year: 2007, vol: 355, page: 28, stat: Journal Article,

Protein kinase C activation inhibits alpha(1D) L-type calcium channel at N-terminal serine 81 phosphorylation site
Baroudi, G; Ou, Y; Ramadan, O; Chahine, M; Boutjdir, M
2006 OCT 31 ;114(18):290-290, Circulation
— id: 69549, year: 2006, vol: 114, page: 290, stat: Journal Article,

Protein kinase C activation inhibits Cav1.3 calcium channel at NH2-terminal serine 81 phosphorylation site
Baroudi, Ghayath; Qu, Yongxia; Ramadan, Omar; Chahine, Mohamed; Boutjdir, Mohamed
2006 Oct;291(4):H1614-H1622, American journal of physiology. Heart & circulatory physiology
The Ca(v)1.3 (alpha(1D)) variant of L-type Ca(2+) channels plays a vital role in the function of neuroendocrine and cardiovascular systems. In this article, we report on the molecular and functional basis of alpha(1D) Ca(2+) channel modulation by protein kinase C (PKC). Specifically, we show that the serine 81 (S81) phosphorylation site at the NH(2)-terminal region plays a critical role in alpha(1D) Ca(2+) channel modulation by PKC. The introduction of a negatively charged residue at position 81, by converting serine to aspartate, mimicked the PKC phosphorylation effect on alpha(1D) Ca(2+) channel. The modulation of alpha(1D) Ca(2+) channel by PKC was prevented by dialyzing cells with a 35-amino acid peptide mimicking the alpha(1D) NH(2)-terminal region comprising S81. In addition, the data revealed that only betaII- and epsilonPKC isozymes are implicated in this regulation. These novel findings have significant implications in the pathophysiology of alpha(1D) Ca(2+) channel and in the development of PKC isozyme-targeted therapeutics
— id: 126660, year: 2006, vol: 291, page: H1614, stat: Journal Article,

The kinetics of spontaneous calcium oscillations and arrhythmogenesis in the in vivo heart during ischemia/reperfusion
Lakireddy, Vikram; Bub, Gil; Baweja, Paramdeep; Syed, Asma; Boutjdir, Mohamed; El-Sherif, Nabil
2006 Jan;3(1):58-66, Heart rhythm
BACKGROUND: The correlation between spontaneous calcium oscillations (S-CaOs) and arrhythmogenesis has been investigated in a number of theoretical and experimental in vitro models. There is an obvious lack of studies that directly investigate how the kinetics of S-CaOs correlates with a specific arrhythmia in the in vivo heart. OBJECTIVES: The purpose of the study is to investigate the correlation between the kinetics of S-CaOs and arrhythmogenesis in the intact heart using an experimental model of ischemia/reperfusion (I/R). METHODS: Perfused Langendorff guinea pig (GP) hearts were subjected to global I/R (10-15 minutes/10-15 minutes). The heart was stained with a voltage-sensitive dye (RH237) and loaded with a Ca2+ indicator (Rhod-2 AM). Membrane voltage (Vm) and intracellular calcium transient (Ca(i)T) were simultaneously recorded with an optical mapping system of two 16 x 16 photodiode arrays. S-CaOs were considered to arise from a localized focal site within the mapped surface when these preceded the associated membrane depolarizations by 2-15 ms. RESULTS: In 135 episodes of ventricular arrhythmias from 28 different GP experiments, 23 were linked to S-CaOs that were considered to arise from or close to the mapped epicardial window. Self-limited or sustained S-CaOs had a cycle length of 130-430 ms and could trigger propagated ventricular depolarizations. Self-limited S-CaOs that followed the basic beat action potential (AP)/Ca(i)T closely resembled phase 3 early afterdepolarizations. Fast S-CaOs could remain confined to a localized site (concealed) or exhibit varying conduction patterns. This could manifest as (1) an isolated premature beat (PB), bigeminal, or trigeminal rhythm; (2) ventricular tachycardia (VT) when a regular 2:1 conduction from the focal site develops; or (3) ventricular fibrillation (VF) when a complex conduction pattern results in wave break and reentrant excitation. CONCLUSIONS: The study examined, for the first time in the intact heart, the correlation between the kinetics of focal S-CaOs during I/R and arrhythmogenesis. S-CaOs may remain concealed or manifest as PBs, VT, or VF. A 'benign looking' PB during I/R may represent 'the tip of the iceberg' of an underlying potentially serious arrhythmic mechanism
— id: 126662, year: 2006, vol: 3, page: 58, stat: Journal Article,

Two dual specificity kinases are preferentially induced by wild-type rather than by oncogenic RAS-P21 in Xenopus oocytes
Qu, Yongxia; Adler, Victor; Chu, Tearina; Platica, Ovidu; Michl, Josef; Pestka, Sidney; Izotova, Lara; Boutjdir, Mohamed; Pincus, Matthew R
2006 ;11:2420-2427, Frontiers in biosciences
In prior studies, we have found that oncogenic ras-p21 protein induces oocyte maturation using pathways that differ from those activated by insulin-induced wild-type ras-p21. Both oncogenic and wild-type ras-p21 require interactions with raf, but unlike oncogenic ras-p21, insulin-activated wild-type ras-p21 does not depend completely on activation of MEK and MAP kinase (MAPK or ERK) on the raf kinase pathway. To determine what raf-dependent but MAPK-independent pathway is activated by wild-type ras-p21, we have analyzed gene expression in oocytes induced to mature either with oncogenic ras-p21 or with insulin using a newly available Xenopus gene array. We find a number of proteins that are preferentially expressed in one or the other system. Of these, two proteins, both dual function kinases, T-Cell Origin Protein Kinase (TOPK) and the nuclear kinase, DYRK1A, are preferentially expressed in the insulin system. Confirming this finding, blots of lysates of oocytes, induced to mature with oncogenic ras-p21 and insulin, with anti-TOPK and anti-DYRK1A show much higher protein expression in the lysates from the insulin-matured oocytes. Neither of these kinases activates or is activated by MAPK and is therefore an attractive candidate for being on a signal transduction pathway that is unique to insulin-activated wild-type ras-p21-induced oocyte maturation
— id: 126661, year: 2006, vol: 11, page: 2420, stat: Journal Article,

Protective role of protein kinase C epsilon activation in ischemia-reperfusion arrhythmia
Yue, Yuankun; Qu, Yongxia; Boutjdir, Mohamed
2006 Oct 13;349(1):432-438, Biochemical & biophysical research communications
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
— id: 69246, year: 2006, vol: 349, page: 432, stat: Journal Article,

Functional interactions of Raf and MEK with Jun-N-terminal kinase (JNK) result in a positive feedback loop on the oncogenic Ras signaling pathway
Adler, Victor; Qu, Yongxia; Smith, Steven J; Izotova, Lara; Pestka, Sidney; Kung, Hsiang-Fu; Lin, Marie; Friedman, Fred K; Chie, Lyndon; Chung, Denise; Boutjdir, Mohamed; Pincus, Matthew R
2005 Aug 16;44(32):10784-10795, Biochemistry
In previous studies we have found that oncogenic (Val 12)-ras-p21 induces Xenopus laevis oocyte maturation that is selectively blocked by two ras-p21 peptides, 35-47, also called PNC-7, that blocks its interaction with raf, and 96-110, also called PNC-2, that blocks its interaction with jun-N-terminal kinase (JNK). Each peptide blocks activation of both JNK and MAP kinase (MAPK or ERK) suggesting interaction between the raf-MEK-ERK and JNK-jun pathways. We further found that dominant negative raf blocks JNK induction of oocyte maturation, again suggesting cross-talk between pathways. In this study, we have undertaken to determine where these points of cross-talk occur. First, we have immunoprecipitated injected Val 12-Ha-ras-p21 from oocytes and found that a complex forms between ras-p21 raf, MEK, MAPK, and JNK. Co-injection of either peptide, but not a control peptide, causes diminished binding of ras-p21, raf, and JNK. Thus, one site of interaction is cooperative binding of Val 12-ras-p21 to raf and JNK. Second, we have injected JNK, c-raf, and MEK into oocytes alone and in the presence of raf and MEK inhibitors and found that JNK activation is independent of the raf-MEK-MAPK pathway but that activated JNK activates raf, allowing for activation of ERK. Furthermore, we have found that constitutively activated MEK activates JNK. We have corroborated these findings in studies with isolated protein components from a human astrocyte (U-251) cell line; that is, JNK phosphorylates raf but not the reverse; MEK phosphorylates JNK but not the reverse. We further have found that JNK does not phosphorylate MAPK and that MAPK does not phosphorylate JNK. The stress-inducing agent, anisomycin, causes activation of JNK, raf, MEK, and ERK in this cell line; activation of JNK is not inhibitable by the MEK inhibitor, U0126, while activation of raf, MEK, and ERK are blocked by this agent. These results suggest that activated JNK can, in turn, activate not only jun but also raf that, in turn, activates MEK that can then cross-activate JNK in a positive feedback loop
— id: 126663, year: 2005, vol: 44, page: 10784, stat: Journal Article,

Contrasting effects of ischemia on the kinetics of membrane voltage and intracellular calcium transient underlie electrical alternans
Lakireddy, Vikram; Baweja, Paramdeep; Syed, Asma; Bub, Gil; Boutjdir, Mohamed; El-Sherif, Nabil
2005 Jan;288(1):H400-H407, American journal of physiology. Heart & circulatory physiology
Repolarization alternans has been considered a strong marker of electrical instability. The objective of this study was to investigate the hypothesis that ischemia-induced contrasting effects on the kinetics of membrane voltage and intracellular calcium transient (Ca(i)T) can explain the vulnerability of the ischemic heart to repolarization alternans. Ischemia-induced changes in action potential (AP) and Ca(i)T resulting in alternans were investigated in perfused Langendorff guinea pig hearts subjected to 10-15 min of global no-flow ischemia followed by 10-15 min of reperfusion. The heart was stained with 100 microl of rhod-2 AM and 25 microl of RH-237, and AP and Ca(i)T were simultaneously recorded with an optical mapping system of two 16 x 16 photodiode arrays. Ischemia was associated with shortening of AP duration (D) but delayed upstroke, broadening of peak, and slowed decay of Ca(i)T resulting in a significant increase of Ca(i)T-D. The changes in APD were spatially heterogeneous in contrast to a more spatially homogeneous lengthening of Ca(i)T-D. Ca(i)T alternans could be consistently induced with the introduction of a shorter cycle when the upstroke of the AP occurred before complete relaxation of the previous Ca(i)T and generated a reduced Ca(i)T. However, alternans of Ca(i)T was not necessarily associated with alternans of APD, and this was correlated with the degree of spatially heterogeneous shortening of APD. Sites with less shortening of APD developed alternans of both Ca(i)T and APD, whereas sites with greater shortening of APD could develop a similar degree of Ca(i)T alternans but slight or no APD alternans. This resulted in significant spatial dispersion of APD. The study shows that the contrasting effects of ischemia on the duration of AP and Ca(i)T and, in particular, on their spatial distribution explain the vulnerability of ischemic heart to alternans and the increased dispersion of repolarization during alternans
— id: 126665, year: 2005, vol: 288, page: H400, stat: Journal Article,

Novel molecular mechanism involving alpha1D (Cav1.3) L-type calcium channel in autoimmune-associated sinus bradycardia
Qu, Yongxia; Baroudi, Ghayath; Yue, Yuankun; Boutjdir, Mohamed
2005 Jun 14;111(23):3034-3041, Circulation
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 alpha1D 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 alpha1D 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 alpha1D Ca channel in the human fetal heart. The effect of positive IgG on alpha1D Ca current (I(Ca-L)) was characterized in heterologous expression systems (tsA201 cells and Xenopus oocytes) because of the unavailability of alpha1D-specific modulators. alpha1D I(Ca-L) activated at negative potentials (between -60 and -50 mV). Positive IgG inhibited alpha1D I(Ca-L) in both expression systems. This inhibition was rescued by a Ca channel activator, Bay K8644. No effect on alpha1D I(Ca-L) was observed with negative IgG and denatured positive IgG. Western blot data showed that positive IgG binds directly to alpha1D Ca channel protein. CONCLUSIONS: The data are the first to demonstrate (1) expression of the alpha1D Ca channel in human fetal heart, (2) inhibition of alpha1D I(Ca-L) by positive IgG, and (3) direct cross-reactivity of positive IgG with the alpha1D Ca channel protein. Given that alpha1D I(Ca-L) activates at voltages within the pacemaker's diastolic depolarization, inhibition of alpha1D I(Ca-L) in part may account for autoimmune-associated sinus bradycardia. In addition, Bay K8644 rescue of alpha1D I(Ca-L) inhibition opens new directions in the development of pharmacotherapeutic approaches in the management of CHB
— id: 62392, year: 2005, vol: 111, page: 3034, stat: Journal Article,

Localization and modulation of {alpha}1D (Cav1.3) L-type Ca channel by protein kinase A
Qu, Yongxia; Baroudi, Ghayath; Yue, Yuankun; El-Sherif, Nabil; Boutjdir, Mohamed
2005 May;288(5):H2123-H2130, American journal of physiology. Heart & circulatory physiology
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
— id: 126664, year: 2005, vol: 288, page: H2123, stat: Journal Article,

Functional basis of sinus bradycardia in congenital heart block
Hu, Keli; Qu, Yongxia; Yue, Yuankun; Boutjdir, Mohamed
2004 Apr 5;94(4):e32-e38, Circulation research
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- (I(Ca.L)) and T- (I(Ca.T)) type Ca2+, delayed rectifier K+ (I(K)), hyperpolarization-activated (I(f)) currents, and action potentials (APs) from young rabbit SA node cells. We demonstrated that positive IgG significantly inhibited both I(Ca.T) and I(Ca.L) and induced sinus bradycardia but did not affect I(f) and I(K). 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 I(Ca.T) and I(Ca.L), two important pacemaker currents in rabbit SA node cells and point to both I(Ca.T) and I(Ca.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
— id: 46210, year: 2004, vol: 94, page: e32, stat: Journal Article,

Modulation of Nav1.7 and Nav1.8 peripheral nerve sodium channels by protein kinase A and protein kinase C
Vijayaragavan, Kausalia; Boutjdir, Mohamed; Chahine, Mohamed
2004 Apr;91(4):1556-1569, Journal of neurophysiology
Voltage-gated Na+ channels (VGSC) are transmembrane proteins that are essential for the initiation and propagation of action potentials in neuronal excitability. Because neurons express a mixture of Na+ channel isoforms and protein kinase C (PKC) isozymes, the nature of which channel is being regulated by which PKC isozyme is not known. We showed that DRG VGSC Nav1.7 (TTX-sensitive) and Nav1.8 (TTX-resistant), expressed in Xenopus oocytes were differentially regulated by protein kinase A (PKA) and PKC isozymes using the two-electrode voltage-clamp method. PKA activation resulted in a dose-dependent potentiation of Nav1.8 currents and an attenuation of Nav1.7 currents. PKA-induced increases (Nav1.8) and decreases (Nav1.7) in peak currents were not associated with shifts in voltage-dependent activation or inactivation. The PKA-mediated increase in Nav1.8 current amplitude was prevented by chloroquine, suggesting that cell trafficking may contribute to the changes in Nav1.8 current amplitudes. A dose-dependent decrease in Nav1.7 and Nav1.8 currents was observed with the PKC activators phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate. PMA induced shifts in the steady-state activation of Nav1.7 and Nav1.8 channels by 6.5 and 14 mV, respectively, in the depolarizing direction. The role of individual PKC isozymes in the regulation of Nav1.7 and Nav1.8 was determined using PKC-isozyme-specific peptide activators and inhibitors. The decrease in the Nav1.8 peak current induced by PMA was prevented by a specific epsilonPKC isozyme peptide antagonist, whereas the PMA effect on Nav1.7 was prevented by epsilonPKC and betaIIPKC peptide inhibitors. The data showed that Nav1.7 and Nav1.8 were differentially modulated by PKA and PKC. This is the first report demonstrating a functional role for epsilonPKC and betaIIPKC in the regulation of Nav1.7 and Nav1.8 Na+ channels. Identification of the particular PKC isozymes(s) that mediate the regulation of Na+ channels is essential for understanding the molecular mechanism involved in neuronal ion channel regulation in normal and pathological conditions
— id: 126667, year: 2004, vol: 91, page: 1556, stat: Journal Article,

Beta- and alpha-adrenergic cross-signaling for L-type Ca current is impaired in transgenic mice with constitutive activation of epsilonPKC
Yue, Yuankun; Qu, Yongxia; Boutjdir, Mohamed
2004 Feb 13;314(3):749-754, Biochemical & biophysical research communications
It is well established that beta-adrenoceptor stimulation activates PKA and alpha(1)-adrenoceptor stimulation activates PKC. In normal ventricular myocytes, acute activation of alpha(1)-adrenoceptors inhibits beta-adrenoceptor stimulated L-type Ca current (I(Ca-L)) and direct activation of epsilonPKC leads to I(Ca-L) inhibition. Because increased PKC activity has been observed chronically in in vivo setting such as failing human heart, we hypothesized that chronic in vivo activation of epsilonPKC alters I(Ca-L) and its response to adrenergic stimulation. Therefore, we investigated the interaction between beta- and alpha(1)-adrenoceptors vis-a-vis I(Ca-L) in myocytes from transgenic mice (TG) with cardiac specific constitutive activation of epsilonPKC (epsilonPKC agonist). Whole-cell I(Ca-L) was recorded from epsilonPKC agonist TG mice and age-matched non-TG (NTG) littermates under: (1) basal condition, (2) beta-adrenoceptor agonist, isoproterenol (ISO), and (3) ISO plus alpha(1)-adrenoceptor agonist, methoxamine. The present results are the first to demonstrate that chronic in vivo activation of epsilonPKC leads to reduced basal I(Ca-L) density. beta-adrenoceptor activation of I(Ca-L) is blunted in epsilonPKC agonist TG mice. alpha-adrenoceptor cross-talk with beta-adrenoceptor signaling pathways vis-a-vis L-type Ca channels is impaired in epsilonPKC agonist TG mice. The diminished response to ISO and methoxamine suggests a protective feedback regulatory mechanism in epsilonPKC agonist TG mice and could be vital in the settings of excessive release of catecholamines during heart failure
— id: 126666, year: 2004, vol: 314, page: 749, stat: Journal Article,

epsilon PKC inhibits human cardiac alpha(1D) (Ca(v)1.3) calcium channels
Baroudi, G; Qu, YX; Yue, YK; Boutjdir, M
2003 OCT 28 ;108(17):239-239, Circulation
— id: 42527, year: 2003, vol: 108, page: 239, stat: Journal Article,

Oncogenic and activated wild-type ras-p21 proteins induce different isoforms of protein kinase C in mitogenic signal transduction
Chie, L; Qu, YX; Chung, D; Boutjdir, M; Pincus, MR
2003 NOV ;22(7-8):625-629, Journal of protein chemistry
We have previously found that the protein kinase C (PKC) inhibitor, CGP 41 251, blocks oncogenic ras-p21 protein- and beta-PKC-induced oocyte maturation, but only weakly inhibits insulin-induced oocyte maturation (which requires activation of wild-type endogenous ras-p21). Because the dose-response curves for inhibition of oncogenic p21- and beta-PKC-induced oocyte maturation by CGP 41 251 superimpose and because the ras-p21-inactivating antibody, Y13-259, does not inhibit beta-PKC-induced oocyte maturation, we concluded that the oncogenic, but not wild-type, protein requires beta-PKC as a downstream target. Because multiple isoforms of PKC exist and several of these, such as epsilon-PKC, have been found to be important on ras signal transduction pathways, we have investigated which PKC isoforms are critical to each ras protein. For this purpose, we used PKC-isoform-specific inhibitors, which have been shown to inhibit selectively the function and translocation of PKC isoforms in vitro and in vivo. Specifically, the peptides KLFIMN, QEVIRN, and EAVSLK
— id: 42575, year: 2003, vol: 22, page: 625, stat: Journal Article,

Localization and modulation of human alpha(1D) (Ca(v)1.3) L-type ca channel by protein kinase a
Qu, YX; Baroudi, G; Yue, Y; Korichneva, I; Boutjdir, M
2003 OCT 28 ;108(17):31-32, Circulation
— id: 42524, year: 2003, vol: 108, page: 31, stat: Journal Article,

alpha(1D) (Ca(v)1.3) L-type Ca channel plays a role in autoimmune-associated sinus bradycardia
Qu, YX; Yue, YK; Baroudi, G; Korichneva, I; Boutjdir, M
2003 OCT 28 ;108(17):360-360, Circulation
— id: 42528, year: 2003, vol: 108, page: 360, stat: Journal Article,

PKC isozyme selective regulation of cloned human cardiac delayed slow rectifier K current
Xiao, Guang-Qian; Mochly-Rosen, Daria; Boutjdir, Mohamed
2003 Jul 11;306(4):1019-1025, Biochemical & biophysical research communications
Delayed rectifying K(+) channel, I(Ks), plays a vital role in normal and arrhythmogenic heart. I(Ks) is modulated by PKC but the identity of which PKC isozymes is involved in this modulation is not known. To dissect the role of individual PKC isozymes in the regulation of I(Ks), human cardiac I(Ks) channel (minK+KvLQT1) was expressed in Xenopus oocytes. Peptide PKC isozyme-specific activator and inhibitors, in addition to the general PKC activator, PMA, were used. Whole-cell I(Ks) was recorded using two-electrode voltage clamp technique. PMA and epsilon PKC specific activator peptide, but not the inactive analog, 4alphaPDD, significantly increased I(Ks). Peptide specific inhibitors for beta(II)PKC, and a general PKC inhibitor, calphostin C antagonized PMA-induced activation of I(Ks). However, control peptide, pentalysine, and specific inhibitor peptide for alphaPKC, beta(I)PKC, deltaPKC, or etaPKC did not alter PMA effect on I(Ks). The present study demonstrates that beta(II)PKC, epsilon PKC but not beta(I)PKC, alphaPKC, deltaPKC, and etaPKC, are involved in PMA-induced activation of the cloned human I(Ks) expressed in Xenopus oocyte. Furthermore, this is the first report to dissect the fine functional role of beta(II)PKC and beta(I)PKC in the regulation of I(Ks). Identification of the particular isozyme(s) that mediates the regulation of I(Ks) channels is of importance for the understanding of the mechanism of ion channel regulation and the development of new therapeutic agents
— id: 126668, year: 2003, vol: 306, page: 1019, stat: Journal Article,

Cardiac 5-HT(4) Serotoninergic Receptors, 52kD SSA/Ro and Autoimmune-Associated Congenital Heart Block
Buyon, Jill P; Clancy, Robert; Di Donato, Francis; Miranda-Carus, M Eugenia; Askanase, Anca D; Garcia, Joanne; Qu, Yongxia; Hu, Keli; Yue, Yuankun; Chan, Edward K L; Boutjdir, Mohamed
2002 Aug;19(1-2):79-79, Journal of autoimmunity
It was recently reported that sera from patients with systemic lupus erythematosus contain antibodies reactive with the second extracellular loop of the serotoninergic 5-HT(4) receptor expressed in the human heart. This antibody response was associated with antibodies to 52kD SSA/Ro, a reactivity prevalent in mothers of children with congenital heart block (CHB). The current study was undertaken to determine whether the 5-HT(4) receptor is a target of the immune response in these mothers. Initial experiments demonstrated mRNA expression of the 5-HT(4) receptor in the human foetal atrium. Electrophysiologic studies established that human foetal atrial cells express functional 5-HT(4) receptors. Sera from 116 mothers enrolled in the Research Registry for Neonatal Lupus, whose children have CHB, were evaluated. Ninety-nine (85%) of these maternal sera contained antibodies to SSA/Ro, 84% of which were reactive with the 52kD SSA/Ro component by immunoblot. None of the 116 sera were reactive with the peptide spanning aa165-185 of the serotoninergic receptor. Rabbit antisera which recognized this peptide did not react with 52kD SSA/Ro or peptide aa365-382 in the C terminus. Although 5-HT(4) receptors are present and functional in the human foetal heart, maternal antibodies to the 5-HT(4) receptor are not associated with the development of CHB
— id: 32693, year: 2002, vol: 19, page: 79, stat: Journal Article,

Cardiac 5-HT4 serotoninergic receptors, 52kD SSA/Ro and autoimmune-associated congenital heart block
Boutjdir, M; Qu, YX; Hu, KL; Di Donato, F; Miranda-Carus, E; Askanase, AD; Garcia, J; Chan, EK; Buyon, JP
2001 OCT 23 ;104(17):752-752, Circulation
— id: 54805, year: 2001, vol: 104, page: 752, stat: Journal Article,

Anti-re-associated sinus bradycardia in newborns - Response
Boutjdir, M; El-Sherif, N; Mazel, JA
2000 SEP 12 ;102(11):E88-E89, Circulation
— id: 54529, year: 2000, vol: 102, page: E88, stat: Journal Article,

mRNA and protein expression of SSA/Ro and SSB/La in human fetal cardiac myocytes cultured using a novel application of the Langendorff procedure
Tseng CE; Miranda E; Di Donato F; Boutjdir M; Rashbaum W; Chan EK; Buyon JP
1999 Feb;45(2):260-269, Pediatric research
Irreversible congenital heart block (CHB) and the transient rash of neonatal lupus are strongly associated with maternal antibodies to SSA/Ro and SSB/La proteins; however, the precise mechanism by which these antibodies mediate organ-specific injury is not yet defined. Culturing of keratinocytes has provided critical insights. Accordingly, successful culturing of human fetal cardiac myocytes at high yield would constitute a powerful tool to directly examine conditions that promote expression of the target autoantigens. To accomplish this aim, fetal cardiac myocytes from 18- to 22-wk abortuses were established in culture using a novel technique in which cells were isolated after perfusion of the aorta with collagenase in a Langendorff apparatus. After preplating to decrease fibroblast contamination, cardiocytes were grown in flasks and slide chambers. Staining with monoclonal anti-sarcomeric alpha-actinin revealed the expected striations typical of cardiac myocytes in 70-90% of the cells after 4 d in culture. Furthermore, the cells were observed to beat at rates varying between 25-75 beats per minute (bpm) after the addition of 1.8 mM CaCl2. An average yield of 45-60 x 10(6) cells was obtained from a 3- to 5-g heart. Cellular localization of SSA/Ro and SSB/La by indirect immunofluorescence and demonstration of mRNA expression by reverse transcriptase polymerase chain reaction supports the feasibility of cultured cardiac myocytes for the study of congenital heart block. In contrast to the increased expression of SSA/Ro reported for keratinocytes, incubation of cultured human cardiac myocytes with either 17beta-estradiol or progesterone did not alter mRNA expression or cellular localization of 48 kD SSB/La, 52 kD SSA/Ro, or 60 kD SSA/Ro. In summary, we describe a novel method to successfully culture human fetal cardiac myocytes that should provide a valuable resource for investigation of the molecular mechanism(s) contributing to the development of congenital heart block. Differential constitutive and estradiol-induced expression of 52 and 60 kD SSA/Ro in human cardiac myocytes compared with keratinocytes may be a factor contributing to the marked discordance of clinically detectable injury in these two target tissues
— id: 57078, year: 1999, vol: 45, page: 260, stat: Journal Article,

Serum and immunoglobulin G from the mother of a child with congenital heart block induce conduction abnormalities and inhibit L-type calcium channels in a rat heart model
Boutjdir M; Chen L; Zhang ZH; Tseng CE; El-Sherif N; Buyon JP
1998 Jul;44(1):11-19, Pediatric research
Although a strong clinical association exists between congenital heart block (CHB) and an immune response to SSA/Ro and SSB/La proteins, a causative role of these antibodies in the pathogenesis is just emerging. In a preliminary report, we have demonstrated that IgG fractions isolated from the sera of mothers whose children have CHB are arrhythmogenic in the human fetal heart. To more precisely define the arrhythmogenic effect of anti-SSA/Ro-SSB/La antibodies, we used the readily available rat heart model to record: 1) ECGs from Langendorff beating hearts; 2) action potentials from atrioventricular (AV) nodal preparations; 3) L-type Ca currents, I(Ca) at the whole-cell and single channel levels; and 4) other currents such as the transient outward K+ current, I(to), the inward rectifier K+ current, I(K1), and the Na+ current, I(Na). Perfusion of hearts with purified IgG (800 microg/mL), isolated from the serum of a mother with SSA/Ro and SSB/La antibodies whose child had CHB, resulted in bradycardia associated with 2:1 AV block. Simultaneous action potentials were recorded from dissected atrial and AV nodal areas of the rat heart. Superfusion of these preparations with the same mother's IgG fraction resulted in 2:1 AV block followed by complete inhibition of AV nodal action potential. Because AV nodal electrogenesis is largely dependent on I(Ca), the effect of these antibodies on I(Ca) was subsequently determined. Superfusion of myocytes with whole serum or purified IgG (80 microg/mL) from the same mother consistently inhibited whole cell I(Ca), ensemble average Ba2+ currents (I(Ba)) and open state probability, p(o), without affecting the channel conductance. IgG had no significant effect on I(to), I(K1), or I(Na). Whole sera and IgG fractions from a healthy mother with no detectable anti-SSA/Ro or SSB/La antibodies did not inhibit I(Ca) or I(Ba). These results demonstrate that IgG containing anti-SSA/Ro and -SSB/La antibodies induces complete AV block in beating hearts and in multicellular preparations, thus implicating a preferential interaction of these autoantibodies with Ca channels and/or associated regulatory proteins. This is consistent with the observed inhibition of Ca channels that may be a critical factor contributing to the pathogenesis of CHB
— id: 12093, year: 1998, vol: 44, page: 11, stat: Journal Article,

Induction of antibodies reactive with SSA/Ro-SSB/La and development of congenital heart block in a murine model
Miranda-Carus ME; Boutjdir M; Tseng CE; DiDonato F; Chan EK; Buyon JP
1998 Dec 1;161(11):5886-5892, Journal of immunology
To correlate the arrhythmogenic effects of maternal autoantibodies with the genesis of congenital heart block, female BALB/c mice were immunized with human recombinant 48-kDa SSB/La, 60-kDa SSA/Ro, 52-kDa SSA/Ro (52alpha), and 52beta (amino acids 169-245 deleted) as well as with murine recombinant 52-kDa SSA/Ro. Control animals received beta-galactosidase or a polypeptide encoded by pET-28 alone. Following primary immunization and two boosters, high titer responses to the respective Ags were established by ELISA, immunoblotting, and immunoprecipitation. Sera from mice immunized with either human 52alpha or 52beta immunoprecipitated murine 52Ro. mRNA and protein expression of 52Ro was demonstrated in the newborn murine heart. A spectrum of atrioventricular nodal conduction abnormalities was identified by electrocardiogram. First-degree block was detected in 7% of 27 pups born to mothers immunized with 48La, 20% of 54 pups born to 60Ro-immunized mothers, 6% of 56 pups born to 52alpha-immunized mothers, 7% of 86 pups born to 52beta-immunized mothers, and 9% of 22 pups born to mothers immunized with murine 52Ro. Advanced conduction abnormalities were only identified in offspring of 52alpha- or 52beta-immunized mice. In the 52alpha group, one pup had complete block and another had second-degree block (Wenckebach type); in the 52beta group, five pups had complete block. Maternal Abs to the primary immunogens were detected in the pups. No control had any conduction abnormalities. This Ab-specific animal model provides strong evidence for a pathogenic role of anti-SSA/Ro-SSB/La Abs, particularly 52Ro, in the development of congenital heart block. The range and frequency of conduction defects suggest that additional factors promote disease expression
— id: 7686, year: 1998, vol: 161, page: 5886, stat: Journal Article,

Induction of antibodies reactive with SSA/Ro-SSB/La and development of congenital heart block in a murine model
Miranda-Carus, ME; Boutjdir, M; Tseng, C; DiDonato, F; Chan, EKL; Buyon, JP
1998 MAR ;46(3):234A-234A, Journal of investigative medicine
— id: 53504, year: 1998, vol: 46, page: 234A, stat: Journal Article,

Arrhythmogenicity of IgG and anti-52-kD SSA/Ro affinity-purified antibodies from mothers of children with congenital heart block
Boutjdir, M; Chen, L; Zhang, Z H; Tseng, C E; DiDonato, F; Rashbaum, W; Morris, A; el-Sherif, N; Buyon, J P
1997 Mar;80(3):354-362, Circulation research
An important advance in the description and understanding of congenital heart block (CHB) came in the 1970s with the observation that mothers of affected infants frequently had autoimmune diseases and, in particular, that many maternal sera contained antibodies to SSA/Ro and SSB/La ribonucleoproteins. Although the molecular biology of the candidate antigens has been extensively defined, the arrhythmogenic and electrophysiological effects of their cognate antibodies on the human fetal heart are unknown. In the present study, we provide evidence that IgG-enriched fractions and anti-52-kD SSA/Ro antibodies affinity-purified from sera of mothers whose children have CHB induce complete atrioventricular (AV) block in the human fetal heart perfused by the Langendorff technique and inhibit L-type Ca2+ currents at the whole-cell and single-channel level. Immunization of female BALB/c mice with recombinant 52-kD SSA/Ro protein generated high-titer antibodies that crossed the placenta during pregnancy and were associated with varying degrees of AV conduction abnormalities, including complete AV block, in the pups. These findings strongly suggest that anti-52-kD SSA/Ro antibodies are causally related to the development of CHB
— id: 73556, year: 1997, vol: 80, page: 354, stat: Journal Article,

Arrhythmogenicity of IgG from mothers of children with congenital heart block
Boutjdir, M; Chen, L; Zhang, ZH; Tseng, C; DiDonato, F; ElSherif, N; Buyon, J
1996 OCT 15 ;94(8):4175-4175, Circulation
— id: 52748, year: 1996, vol: 94, page: 4175, stat: Journal Article,

IgG and affinity purified anti-52kD SSA/Ro antibodies from mothers of children with congenital heart block induce conduction defects and inhibit Ca channels in the human fetal heart
Boutjdir, M; Chen, L; Zhang, ZH; Tseng, C; DiDonato, F; Rashburn, W; Morris, W; ElSherif, N; Buyon, J
1996 OCT 15 ;94(8):3471-3471, Circulation
— id: 52747, year: 1996, vol: 94, page: 3471, stat: Journal Article,

ELECTROPHYSIOLOGIC CHARACTERIZATION OF PURIFIED IGG FROM A MOTHER WHOSE CHILD HAS CONGENITAL HEART-BLOCK (CHB) ON L-TYPE CALCIUM CURRENTS (I-CA)
BOUTJDIR, M; ZHANG, ZH; CHEN, L; ELSHERIF, N; TSENG, CE; DIDONATO, F; RASHBAUM, W; BUYON, JP
1995 SEP ;38(9):466-466, Arthritis & rheumatism
— id: 86695, year: 1995, vol: 38, page: 466, stat: Journal Article,