Martin B. Rook

Compound Heterozygosity for Mutations (W156X and R225W) in SCN5A Associated With Severe Cardiac Conduction Disturbances and Degenerative Changes in the Conduction System

Abstract— Cardiac conduction defects associate with mutations in SCN5A, the gene encoding the cardiac Na+ channel. In the present study, we characterized a family in which the proband was born in severe distress with irregular wide complex tachycardia. His older sister died at 1 year of age from severe conduction disease with similarly widened QRS-complexes. Mutational analysis of SCN5A in the proband demonstrated compound heterozygosity for a nonsense mutation (W156X), inherited from the father, and a missense mutation (R225W), inherited from the mother. Genotyping on DNA extracted from tissue from the deceased sibling revealed the same SCN5A genotype. Injection of cRNA encoding the W156X mutation in Xenopus oocytes did not produce any current. The R225W substitution neutralizes the third Arg residue within the voltage-sensing segment of domain I. Expression studies showed that this mutation leads to a severe reduction in INa and is also associated with gating changes. Histological examination of the heart from the deceased sibling revealed changes consistent with a dilated type of cardiomyopathy and severe degenerative abnormalities of the specialized conduction system. The occurrence of compound heterozygosity for these two mutations implies that the proband carries solely severely dysfunctional cardiac Na+ channels. This explains his severe phenotype and that of his deceased sister who had been a carrier of the same genotype. The morphological changes within the heart of the deceased sibling may have occurred secondary to the Na+ channel abnormality and contributed to the severity of the disorder in this individual.

Human SCN5A gene mutations alter cardiac sodium channel kinetics and are associated with the Brugada syndrome.

BACKGROUND Primary dysrhythmias other than those associated with the long QT syndrome, are increasingly recognized. One of these are represented by patients with a history of resuscitation from cardiac arrest but without any structural heart disease. These patients exhibit a distinct electrocardiographic (ECG) pattern consisting of a persistent ST-segment elevation in the right precordial leads often but not always accompanied by a right bundle branch block (Brugada syndrome). This syndrome is associated with a high mortality rate and has been shown to display familial occurrence. METHODS AND RESULTS Pharmacological sodium channel blockade elicits or worsens the electrocardiographic features associated with this syndrome. Hence, a candidate gene approach directed towards SCN5A, the gene encoding the alpha-subunit of the cardiac sodium channel, was followed in six affected individuals. In two patients missense mutations were identified in the coding region of the gene: R1512W in the DIII-DIV cytoplasmic linker and A1924T in the C-terminal cytoplasmic domain. In two other patients mutations were detected near intron/exon junctions. To assess the functional consequences of the R1512W and A1924T mutations, wild-type and mutant sodium channel proteins were expressed in Xenopus oocytes. Both missense mutations affected channel function, most notably a 4-5 mV negative voltage shift of the steady-state activation and inactivation curves in R1512W and a 9 mV negative voltage shift of the steady-state activation curve in A1924T, measured at 22 degrees C. Recovery from inactivation was slightly prolonged for R1512W channels. The time dependent kinetics of activation and inactivation at -20 mV were not significantly affected by either mutation. CONCLUSIONS Two SCN5A mutations associated with the Brugada syndrome, significantly affect cardiac sodium channel characteristics. The alterations seem to be associated with an increase in inward sodium current during the action potential upstroke.

Cardiac sodium channel and inherited arrhythmia syndromes

Time for primary review 32 days. The voltage-gated cardiac sodium channel is responsible for the generation of the rapid upstroke of the myocardial action potential and thereby plays a central role in excitability of myocardial cells. In addition, since the action potential upstroke velocity — in conjunction with the extent of intercellular communication via gap junctions [1] — also determines impulse conduction velocity in cardiac tissue, this channel also plays a vital role in impulse propagation. Mutations in the gene encoding this channel ( SCN 5 A ; Section 2.1) have been linked to three forms of primary electrical disease — the long QT syndrome (LQTS) [2], the Brugada syndrome (BS) [3] and cardiac conduction defects [4]. The elucidation of the pathophysiological mechanisms of these mutant Na+ channels would ultimately enable more specific pharmacological intervention in the management of these syndromes and other related arrhythmias. The cardiac Na+ channel (see Ref. [5] for review) is a member of the voltage-dependent family of Na+ channels (see Ref. [6] for review). These channels consist of heteromeric assemblies of an α-subunit, the pore-forming component, the function of which is modulated by association with one or two ancillary β-subunits. ### 2.1 α-Subunit The human cardiac Na+ channel α-subunit is a heavily glycosylated protein of ∼260 kDa consisting of 2016 amino acid residues [7]. It is encoded by the SCN 5 A gene [8], which is located on chromosome 3p21 [9]. At least 11 other different genes that encode highly homologous sodium channel α-subunit isoforms expressed in the heart, muscle and nervous system have been hitherto identified in man (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide). They display a modular architecture (Fig. 1), which consists of four internally homologous domains (DI–DIV) each made up of six transmembrane segments (S1–S6). The interdomain linkers and the N- and … * Corresponding author. Tel.: +31-20-566-3265; fax: +31-20-697-5458 a.a.wilde{at}amc.uva.nl

Biology of cardiac sodium channel Nav1.5 expression

Na(v)1.5, the pore forming α-subunit of the voltage-dependent cardiac Na(+) channel, is an integral membrane protein involved in the initiation and conduction of action potentials. Mutations in the gene-encoding Na(v)1.5, SCN5A, have been associated with a variety of arrhythmic disorders, including long QT, Brugada, and sick sinus syndromes as well as progressive cardiac conduction defect and atrial standstill. Moreover, alterations in the Na(v)1.5 expression level and/or sodium current density have been frequently noticed in acquired cardiac disorders, such as heart failure. The molecular mechanisms underlying these alterations are poorly understood, but are considered essential for conception of arrhythmogenesis and the development of therapeutic strategies for prevention or treatment of arrhythmias. The unravelling of such mechanisms requires critical molecular insight into the biology of Na(v)1.5 expression and function. Therefore, the aim of this review is to provide an up-to-date account of molecular determinants of normal Na(v)1.5 expression and function. The parts of the Na(v)1.5 life cycle that are discussed include (i) regulatory aspects of the SCN5A gene and transcript structure, (ii) the nature, molecular determinants, and functional consequences of Na(v)1.5 post-translational modifications, and (iii) the role of Na(v)1.5 interacting proteins in cellular trafficking. The reviewed studies have provided valuable information on how the Na(v)1.5 expression level, localization, and biophysical properties are regulated, but also revealed that our understanding of the underlying mechanisms is still limited.

Dominant missense mutations in ABCC9 cause Cantú syndrome

Cantú syndrome is characterized by congenital hypertrichosis, distinctive facial features, osteochondrodysplasia and cardiac defects. By using family-based exome sequencing, we identified a de novo mutation in ABCC9. Subsequently, we discovered novel dominant missense mutations in ABCC9 in 14 of the 16 individuals with Cantú syndrome examined. The ABCC9 protein is part of an ATP-dependent potassium (KATP) channel that couples the metabolic state of a cell with its electrical activity. All mutations altered amino acids in or close to the transmembrane domains of ABCC9. Using electrophysiological measurements, we show that mutations in ABCC9 reduce the ATP-mediated potassium channel inhibition, resulting in channel opening. Moreover, similarities between the phenotype of individuals with Cantú syndrome and side effects from the KATP channel agonist minoxidil indicate that the mutations in ABCC9 result in channel opening. Given the availability of ABCC9 antagonists, our findings may have direct implications for the treatment of individuals with Cantú syndrome.

P19 embryonal carcinoma cells: a suitable model system for cardiac electrophysiological differentiation at the molecular and functional level

Objective: Murine P19 embryonal carcinoma (EC) cells can differentiate into spontaneously beating cardiomyocytes in vitro and have revealed important insight into the early molecular processes of cardiomyocyte differentiation. We assessed the suitability of the P19 cell model for studying cardiac ion channel regulation at the molecular and functional level. Methods: P19 cells were induced to differentiate towards cardiomyocytes. mRNAs for cardiac markers and ion channels were determined by RT-PCR at six timepoints during the differentiation process. Action potentials and individual ion currents were measured by whole cell patch clamp. Results: Ion channel mRNA expression of several channels is temporally regulated during differentiation, while others show little or no regulation. L-type calcium and transient outward channels are expressed from very early on, while sodium and delayed and inward rectifier channels are upregulated at somewhat later stages during differentiation, which mirrors the in vivo murine cardiomyocyte differentiation during embryogenesis. Spontaneous cardiomyocyte action potentials exhibit a low upstroke velocity, which often can be enhanced by hyperpolarizing the cells, hence activating thusfar dormant ion channels to contribute to the action potential upstroke. Action potential duration decreases considerably during the differentiation of spontaneously beating cells. In late stages, non-beating myocytes can be found which only generate action potentials upon electrical stimulation. Their shape is comparable to neonatal/juvenile ventricular mouse myocytes in culture. Finally, we show that P19-derived cardiomyocytes display a very complete set of functional ion channels. Conclusion: P19 cells represent a powerful model to study the regulation of myocardial electrophysiological differentiation at the molecular and functional level.

Na+ channel mutation leading to loss of function and non-progressive cardiac conduction defects

BACKGROUND We previously described a Dutch family in which congenital cardiac conduction disorder has clinically been identified. The ECG of the index patient showed a first-degree AV block associated with extensive ventricular conduction delay. Sequencing of the SCN5A locus coding for the human cardiac Na+ channel revealed a single nucleotide deletion at position 5280, resulting in a frame-shift in the sequence coding for the pore region of domain IV and a premature stop codon at the C-terminus. METHODS AND RESULTS Wild type and mutant Na+ channel proteins were expressed in Xenopus laevis oocytes and in mammalian cells. Voltage clamp experiments demonstrated the presence of fast activating and inactivating inward currents in cells expressing the wild type channel alone or in combination with the beta1 subinut (SCN1B). In contrast, cells expressing the mutant channels did not show any activation of inward current with or without the beta1 subunit. Culturing transfected cells at 25 degrees C did not restore the Na+ channel activity of the mutant protein. Transient expression of WT and mutant Na+ channels in the form of GFP fusion proteins in COS-7 cells indicated protein expression in the cytosol. But in contrast to WT channels were not associated with the plasma membrane. CONCLUSIONS The SCN5A/5280delG mutation results in the translation into non-function channel proteins that do not reach the plasma membrane. This could explain the cardiac conduction defects in patients carrying the mutation.

Ragged spiking of free calcium in ADP‐stimulated human platelets: regulation of puff‐like calcium signals in vitro and ex vivo

1 Human platelets respond to agonists of G protein (Gq)‐coupled receptors by generating an irregular pattern of spiking changes in cytosolic Ca2+ ([Ca2+]i). We have investigated the ADP‐induced Ca2+ responses of single, Fluo‐3‐loaded platelets in the presence or absence of autologous plasma or whole blood under flow conditions. 2 In plasma‐free platelets, incubated in buffer medium, baseline separated [Ca2+]i peaks always consisted of a rapid rising phase (median time 0.8 s) which was abruptly followed by a slower, mono‐exponential decay phase. The decay constant differed from platelet to platelet, ranging from 0.23 ± 0.02 to 0.63 ± 0.03 s−1 (mean ±s.e.m., n = 3–5), and was used to identify individual Ca2+ release events and to determine the Ca2+ fluxes of the events. 3 Confocal, high‐frequency measurements of adherent, spread platelets (diameter 3‐5 μm) indicated that different optical regions had simultaneous patterns of both low‐ and high‐amplitude Ca2+ release events. 4 With or without plasma or flowing blood, the ADP‐induced Ca2+ signals in platelets had the characteristics of irregular Ca2+ puffs as well as more regular Ca2+ oscillations. Individual [Ca2+]i peaks varied in amplitude and peak‐to‐peak interval, as observed for separated Ca2+ puffs within larger cells. On the other hand, the peaks appeared to group into periods of ragged, shorter‐interval Ca2+ release events with little integration, which were alternated with longer‐interval events. 5 We conclude that the spiking Ca2+ signal generated in these small cells has the characteristics of a ‘poor’ oscillator with an irregular frequency being reactivated from period to period. This platelet signal appears to be similar in an environment of non‐physiological buffer medium and in flowing, whole blood.

Comparison of the IKr blockers moxifloxacin, dofetilide and E‐4031 in five screening models of pro‐arrhythmia reveals lack of specificity of isolated cardiomyocytes

Drug development requires the testing of new chemical entities for adverse effects. For cardiac safety screening, improved assays are urgently needed. Isolated adult cardiomyocytes (CM) and human embryonic stem cell‐derived cardiomyocytes (hESC‐CM) could be used to identify pro‐arrhythmic compounds. In the present study, five assays were employed to investigate their sensitivity and specificity for evaluating the pro‐arrhythmic properties of IKr blockers, using moxifloxacin (safe compound) and dofetilide or E‐4031 (unsafe compounds).

Fever-induced QTc prolongation and ventricular arrhythmias in individuals with type 2 congenital long QT syndrome

Type 2 congenital long QT syndrome (LQT-2) is linked to mutations in the human ether a-go-go-related gene (HERG) and is characterized by rate-corrected QT interval (QTc) prolongation, ventricular arrhythmias, syncope, and sudden death. Recognized triggers of these cardiac events include emotional and acoustic stimuli. Here we investigated the repeated occurrence of fever-induced polymorphic ventricular tachycardia and ventricular fibrillation in 2 LQT-2 patients with A558P missense mutation in HERG. ECG analysis showed increased QTc with fever in both patients. WT, A558P, and WT+A558P HERG were expressed heterologously in HEK293 cells and were studied using biochemical and electrophysiological techniques. A558P proteins showed a trafficking-deficient phenotype. WT+A558P coexpression caused a dominant-negative effect, selectively accelerated the rate of channel inactivation, and reduced the temperature-dependent increase in the WT current. Thus, the WT+A558P current did not increase to the same extent as the WT current, leading to larger current density differences at higher temperatures. A similar temperature-dependent phenotype was seen for coexpression of the trafficking-deficient LQT-2 F640V mutation. We postulate that the weak increase in the HERG current density in WT-mutant coassembled channels contributes to the development of QTc prolongation and arrhythmias at febrile temperatures and suggest that fever is a potential trigger of life-threatening arrhythmias in LQT-2 patients.