Ghayath Baroudi

Electrophysiological characterization of SCN5A mutations causing long QT (E1784K) and Brugada (R1512W and R1432G) syndromes

UNLABELLED Familial long QT syndrome (LQTS) and Brugada syndrome are two distinct human hereditary cardiac diseases known to cause ventricular tachyarrhythmias (torsade de pointes) and idiopathic ventricular fibrillation, respectively, which can both lead to sudden death. OBJECTIVE In this study we have identified and electrophysiologically characterized, in patients having either LQTS or Brugada syndrome, three mutations in SCN5A (a cardiac sodium channel gene). METHOD The mutant channels were expressed in a mammalian expression system and studied by means of the patch clamp technique. RESULTS The R1512W mutation found in our first patient diagnosed with Brugada syndrome produced a slowing of both inactivation and recovery from inactivation. The R4132G mutation found in our second patient who also presented Brugada syndrome, resulted in no measurable sodium currents. Both Brugada syndrome patients showed ST segment elevation and right bundle-branch block, and had experienced syncopes. The E1784K mutation found in the LQTS showed a persistent inward sodium current, a hyperpolarized shift of the steady-sate inactivation and a faster recovery from inactivation. CONCLUSION The different clinical manifestations of these three mutations most probably originate from the distinct electrophysiological abnormalities of the mutant cardiac sodium channels reported in this study.

A newly characterized SCN5A mutation underlying Brugada syndrome unmasked by hyperthermia

Febrile illness has been rarely reported to modulate ST segment elevation in right precordial leads on ECG or even precipitate ventricular fibrillation in patients with Brugada syndrome. We report the case of a patient whose Brugada ECG pattern was unmasked by hyperthermia secondary to acute cholangitis. Serial ECGs showed progressive attenuation of ST segment elevation as body temperature gradually returned to normal. Structural heart disease was ruled out. Intravenous flecainide injection reproduced a less remarkable ST segment elevation. Genetic screening demonstrated a single amino acid substitution (H681P) in the SCN5A gene, thus confirming the diagnosis of Brugada syndrome. In vitro expression of this newly characterized genetic defect revealed novel biophysical abnormalities consisting of a shift in both steady‐state activation and inactivation, resulting in a 60% reduction of sodium window current. Thus, SCN5A‐H681P mutation induces a significant loss of transmembrane current and is clinically associated with a pathologic phenotype that is elicited by hyperthermia. Overall the observed clinical features are in agreement with previous observations and strongly suggest that fever may be an environmental modifier among Brugada syndrome patients with a detrimental (and possibly arrhythmogenic) effect on cardiac repolarization. (J Cardiovasc Electrophysiol, Vol. 14, pp. 407‐411, April 2003)

SCN5A mutation (T1620M) causing Brugada syndrome exhibits different phenotypes when expressed in Xenopus oocytes and mammalian cells

Brugada syndrome is a hereditary cardiac disease causing abnormal ST segment elevation in the ECG, right bundle branch block, ventricular fibrillation and sudden death. In this study we characterized a new mutation in the SCN5A gene (T1620M), causing the Brugada syndrome. The mutated channels were expressed in both Xenopus leavis oocytes and in mammalian tsA201 cells with and without the β‐subunit and studied using the patch clamp technique. Opposite phenotypes were observed depending on the expression system. T1620M mutation led to a faster recovery from inactivation and a shift of steady‐state inactivation to more positive voltages when expressed in Xenopus oocytes. However, using the mammalian expression system no effect on steady‐state inactivation was observed, but this mutation led to a slower recovery from inactivation. Our finding supports the idea that the slower recovery from inactivation of the cardiac sodium channels seen in our mammalian expression system could decrease the density of sodium channels during the cardiac cycle explaining the in vivo arrhythmogenesis in patients with Brugada syndrome.

Biophysical phenotypes of SCN5A mutations causing long QT and Brugada syndromes.

Long QT and Brugada syndromes are two hereditary cardiac diseases. Brugada syndrome has so far been associated with only one gene, SCN5A, which encodes the cardiac sodium channel. However, in long QT syndrome (LQTS) at least six genes, including the SCN5A, are implicated. The substitution (D1790G) causes LQTS and the insertion (D1795) induces both LQTS and Brugada syndromes in carrier patients. hH1/insD1795 and hH1/D1790G mutant channels were expressed in the tsA201 human cell line and characterized using the patch clamp technique in whole‐cell configuration. Our data revealed a persistent inward sodium current of about 6% at −30 mV for both D1790G and insD1795, and a reduction of 62% of channel expression for the insD1795. Moreover, a shift of steady‐state inactivation curve in both mutants was also observed. Our findings uphold the idea that LQT3 is related to a persistent sodium current whereas reduction in the expression level of cardiac sodium channels is one of the biophysical characteristics of Brugada syndrome.

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.

Connexin 43 Hemichannels and Pharmacotherapy of Myocardial Ischemia Injury

Connexin (Cx) is the basic unit in the composition of gap junction channels but also exist in non-junctional unapposed hemichannels (Hc). The gap junction channels are formed by apposition of two hexameric CxHc from adjacent cells and play an essential role in cardiac function by allowing conduction of electrical impulse and exchange of biologically important molecules between myocytes. While most CxHc are engaged in gap junction formation, some unapposed Hc are present in association with various organelles (mitochondria, ER, etc.) but also in sarcolemma where they connect the intracellular and extracellular spaces. Recent evidence indicates that unapposed Hc in the plasma membrane perform functions different from those achieved by gap junction channels, mainly by providing pathways between the cytosol and the extracellular space allowing movement of ions and other small metabolites (Bennett et al., 2003; Goodenough & Paul, 2003), release of ATP and NAD+ (Bruzzone et al., 2001; Cotrina et al., 1998), regulation of cell volume (Quist et al., 2000), and the activation of survival pathways (Plotkin et al., 2002). These Hc are therefore believed to play a prominent role in cellular ion homoeostasis and signalling.

Atrial fibrillation: molecular biology has yet to impact management.

Atrial Fibrillation (AF) is the most common sustained arrhythmia in clinical practice, and its incidence increases significantly with age.1 It is a major etiologic factor in stroke in the elderly, and it increases cardiovascular mortality, especially among patients with congestive heart failure. Atrial remodeling refers to the changes that occur in the atria following rapid atrial pacing or atrial tachyarrhythmias (atrial tachycardia, atrial flutter, and self-terminating episodes of AF) and result in progressive increase in the susceptibility to AF. Atrial remodeling also can develop in the presence of heart failure, which is known to be associated with increased incidence of AF. Atrial remodeling has several important clinical implications. It is purported to be the mechanism by which paroxysmal AF tends to become persistent. It may explain the high incidence of recurrence of AF that occurs early after cardioversion. More importantly, it may be the main reason why long-term AF is resistant to pharmacologic cardioversion. Atrial remodeling has been classified into electrical, contractile, and anatomic.2 These changes are the result of activation of specific signal transduction pathways that induce alterations in the molecular, ionic, electrical, and structural characteristics of atrial myocytes and intracellular matrix. Although considerable progress has been made over the past few years in assessing the molecular basis of the pathogenesis of AF, the mechanistic implication of genes alteration underlying AF is not yet completely understood. In this issue of the Journal, Lai et al.3 set out to investigate the genetic changes in a porcine model of AF induced by long-term rapid atrial pacing. Using cDNA microarray and two-dimensional protein electrophoresis techniques, the authors reported significant changes in mRNA levels in 387 genes in the atrium. The most prominent change was an isoform shift in the MCL-2 message and protein with an increase of the ratio MLC-2V/MLC-2A compared to sinus rhythm. The linkage that the study provided between reprogramming of MCL-2 isoform composition and AF is especially convincing, given that the authors have shown changes in MLC-2 isoforms at both mRNA and proteins levels. The changes in MLC-2 is of particular interest in AF because it has been demonstrated that ventricular MIC-2 isoform switch enhances atrial cardiomyocyte to calcium sensitivity of the myofilament and/or facilitates more effective actin-myosin