Guillermo J. Pérez

Ionic and Cellular Basis for the Predominance of the Brugada Syndrome Phenotype in Males

Background—The Brugada syndrome displays an autosomal dominant mode of transmission with low penetrance. Despite equal genetic transmission of the disease, the clinical phenotype is 8 to 10 times more prevalent in males than in females. The basis for this intriguing sex-related distinction is unknown. The present study tests the hypothesis that the disparity in expression of the Brugada phenotype is a result of a more prominent Ito-mediated action potential notch in the right ventricular (RV) epicardium of males versus females. Methods and Results—We studied epicardial tissue slices, arterially perfused wedge preparations, and dissociated epicardial myocytes isolated from male and female canine hearts. RV epicardium action potential phase 1 amplitude was 64.8±2.0% of that of phase 2 in males compared with 73.8±4.4% in females (P <0.05) at a cycle length of 2000 ms. Ito density was 26% smaller and time constant for inactivation 17% smaller at +40 mV in female versus male RV epicardial cells (P <0.05). The other functional characteristics of Ito, including the voltage dependence of inactivation and time course of reactivation, were no different between the sexes. Pinacidil caused loss of action potential dome in male, but not female, RV epicardial tissue slices. Terfenadine (5 &mgr;mol/L) induced phase 2 reentry in 6 of 7 male but only 2 of 7 female arterially perfused wedge preparations. Two of 6 male and 1 of 2 female preparations developed polymorphic ventricular tachycardia/ventricular fibrillation. Conclusions—Our results suggest that the predominance of the Brugada phenotype in males is a result of the presence of a more prominent Ito in males versus females.

Electrophysiologic properties and antiarrhythmic actions of a novel antianginal agent.

Ranolazine is a novel antianginal agent capable of producing anti-ischemic effects at plasma concentrations of 2 to 6 μM without a significant reduction of heart rate or blood pressure. This review summarizes the electrophysiologic properties of ranolazine. Ranolazine significantly blocks IKr (IC50 = 12 μM), late INa, late ICa, peak ICa, INa-Ca (IC50 = 5.9, 50, 296, and 91 μM, respectively) and IKs (17% at 30,uM), but causes little or no inhibition of Ito or IKl. In left ventricular tissue and wedge preparations, ranolazine produces a concentration-dependent prolongation of action potential duration (APD) in epicardium, but abbreviation of APD of M cells, leading to either no change or a reduction in transmural dispersion of repolarization (TDR). The result is a modest prolongation of the QT interval. Prolongation of APD and QT by ranolazine is fundamentally different from that of other drugs that block IKr and induce torsade de pointes in that APD prolongation is rate-independent (ie, does not display reverse rate-dependent prolongation of APD) and is not associated with early afterdepolarizations, triggered activity, increased spatial dispersion of repolarization, or polymorphic ventricular tachycardia. Torsade de pointes arrhythmias were not observed spontaneously nor could they be induced with programmed electrical stimulation in the presence of ranolazine at concentrations as high as 100 μM. Indeed, ranolazine was found to possess significant antiarrhythmic activity, acting to suppress the arrhythmogenic effects of other QT-prolonging drugs. Ranolazine produces ion channel effects similar to those observed after chronic exposure to amiodarone (reduced late INa, IKs, IKS, and ICa). Ranolazines actions to reduce TDR and suppress early afterdepolarization suggest that in addition to its anti-anginal actions, the drug possesses antiarrhythmic activity.

Genetics and cardiac channelopathies.

Abstract: Sudden cardiac death is a major contributor to mortality in industrialized nations; in fact, it is the cause of more deaths than acquired immune deficiency syndrome, lung and breast cancer, and stroke together. Frequently, the autopsy becomes the principal diagnostic tool because macroscopic and microscopic analyses reveal the underlying cause of death. However, a significant number of sudden cardiac deaths remain unexplained. These cases are referred to as “natural” or arrhythmogenic. In the young, in up to 50% of sudden cardiac death cases, sudden death is the first and only clinical manifestation of an inherited cardiac disease that had remained undetected by conventional clinical investigations. To improve diagnosis, genetic testing has recently been added to these clinical tools. During the last two decades, there has been considerable progress in the understanding about genetics of sudden cardiac death. With that new information, the probands and their family members can make an informed decision regarding their care and know whether and to what extent they are at risk of suffering from the disease. Thus, genetic technology and expertise have become essential for the diagnosis of some forms of inherited cardiac diseases and to provide a basis for subsequent prevention strategies. This review focuses on recent advances in the understanding of cardiopathies owing to genetic investigations.

A Missense Mutation in the Sodium Channel β2 Subunit Reveals SCN2B as a New Candidate Gene for Brugada Syndrome

Brugada Syndrome (BrS) is a familial disease associated with sudden cardiac death. A 20%–25% of BrS patients carry genetic defects that cause loss‐of‐function of the voltage‐gated cardiac sodium channel. Thus, 70%–75% of patients remain without a genetic diagnosis. In this work, we identified a novel missense mutation (p.Asp211Gly) in the sodium β2 subunit encoded by SCN2B, in a woman diagnosed with BrS. We studied the sodium current (INa) from cells coexpressing Nav1.5 and wild‐type (β2WT) or mutant (β2D211G) β2 subunits. Our electrophysiological analysis showed a 39.4% reduction in INa density when Nav1.5 was coexpressed with the β2D211G. Single channel analysis showed that the mutation did not affect the Nav1.5 unitary channel conductance. Instead, protein membrane detection experiments suggested that β2D211G decreases Nav1.5 cell surface expression. The effect of the mutant β2 subunit on the INa strongly suggests that SCN2B is a new candidate gene associated with BrS.

A Common Single Nucleotide Polymorphism Can Exacerbate Long-QT Type 2 Syndrome Leading to Sudden Infant Death

Background—Identification of infants at risk for sudden arrhythmic death remains one of the leading challenges of modern medicine. We present a family in which a common polymorphism (single nucleotide polymorphism) inherited from the father, combined with a stop codon mutation inherited from the mother (both asymptomatic), led to 2 cases of sudden infant death. Methods and Results—KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, CACNA1c, CACNB2b, and KCNJ2 genes were amplified and analyzed by direct sequencing. Functional electrophysiological studies were performed with the single nucleotide polymorphism and mutation expressed singly and in combination in Chinese ovary (CHO-K1) and COS-1 cells. An asymptomatic woman presenting after the death of her 2-day-old infant and spontaneous abortion of a second baby in the first trimester was referred for genetic analysis. The newborn infant had nearly incessant ventricular tachycardia while in utero and a prolonged QTc (560 ms). The mother was asymptomatic but displayed a prolonged QTc. Genetic screening of the mother revealed a heterozygous nonsense mutation (P926AfsX14) in KCNH2, predicting a stop codon. The father was asymptomatic with a normal QTc but had a heterozygous polymorphism (K897T) in KCNH2. The baby who died at 2 days of age and the aborted fetus inherited both K897T and P926AfsX14. Heterologous coexpression of K897T and P926AfsX14 led to loss of function of HERG current much greater than expression of K897T or P926AfsX14 alone. Conclusions—Our data suggest that a common polymorphism (K897T) can markedly accentuate the loss of function of mildly defective HERG channels, leading to long-QT syndrome–mediated arrhythmias and sudden infant death.

Protein arginine methyl transferases-3 and -5 increase cell surface expression of cardiac sodium channel

The α‐subunit of the cardiac voltage‐gated sodium channel (NaV1.5) plays a central role in cardiomyocyte excitability. We have recently reported that NaV1.5 is post‐translationally modified by arginine methylation. Here, we aimed to identify the enzymes that methylate NaV1.5, and to describe the role of arginine methylation on NaV1.5 function. Our results show that protein arginine methyl transferase (PRMT)‐3 and ‐5 methylate NaV1.5 in vitro, interact with NaV1.5 in human embryonic kidney (HEK) cells, and increase NaV1.5 current density by enhancing NaV1.5 cell surface expression. Our observations are the first evidence of regulation of a voltage‐gated ion channel, including calcium, potassium, sodium and TRP channels, by arginine methylation.

A missense mutation in the sodium channel β1b subunit reveals SCN1B as a susceptibility gene underlying long QT syndrome

BACKGROUNDnLong QT syndrome (LQTS) is associated with sudden cardiac death and the prolongation of the QT interval on the electrocardiogram. A comprehensive screening of all genes previously associated with this disease leaves 30% of the patients without a genetic diagnosis. Pathogenic mutations in the sodium channel β subunits have been associated with cardiac channelopathies, including SCN4B mutations in LQTS.nnnOBJECTIVEnTo evaluate the role of mutations in the sodium channel β subunits in LQTS.nnnMETHODSnWe screened for mutations in the genes encoding the 5 sodium β subunits (SCN1B isoforms a and b, SCN2B, SCN3B, and SCN4B) from 30 nonrelated patients who were clinically diagnosed with LQTS without mutations in common LQTS-related genes. We used the patch-clamp technique to study the properties of sodium currents and the action potential duration in human embryonic kidney and HL-1 cells, respectively, in the presence of β1b subunits.nnnRESULTSnThe genetic screening revealed a novel mutation in the SCN1Bb gene (β1bP213T) in an 8-year-old boy. Our electrophysiological analysis revealed that β1bP213T increases late sodium current. In addition, β1bP213T subtly altered Nav1.5 function by shifting the window current, accelerating recovery from inactivation, and decreasing the slow inactivation rate. Moreover, experiments using HL-1 cells revealed that the action potential duration significantly increases when the mutant β1b was overexpressed compared with β1bWT.nnnCONCLUSIONnThese data revealed SCN1Bb as a susceptibility gene responsible for LQTS, highlighting the importance of continuing the search for new genes and mechanisms to decrease the percentage of patients with LQTS remaining without genetic diagnosis.

Clinical and molecular characterization of a cardiac ryanodine receptor founder mutation causing catecholaminergic polymorphic ventricular tachycardia.

BACKGROUNDnCatecholaminergic polymorphic ventricular tachycardia (CPVT) is a difficult-to-diagnose cause of sudden cardiac death (SCD). We identified a family of 1400 individuals with multiple cases of CPVT, including 36 SCDs during youth.nnnOBJECTIVESnWe sought to identify the genetic cause of CPVT in this family, to preventively treat and clinically characterize the mutation-positive individuals, and to functionally characterize the pathogenic mechanisms of the mutation.nnnMETHODSnGenetic testing was performed for 1404 relatives. Mutation-positive individuals were preventively treated with β-blockers and clinically characterized with a serial exercise treadmill test (ETT) and Holter monitoring. In vitro functional studies included caffeine sensitivity and store overload-induced calcium release activity of the mutant channel in HEK293 cells.nnnRESULTSnWe identified the p.G357S_RyR2 mutation, in the cardiac ryanodine receptor, in 179 family members and in 6 SCD cases. No SCD was observed among treated mutation-positive individuals over a median follow-up of 37 months; however, 3 relatives who had refused genetic testing (confirmed mutation-positive individuals) experienced SCD. Holter monitoring did not provide relevant information for CPVT diagnosis. One single ETT was unable to detect complex cardiac arrhythmias in 72% of mutation-positive individuals, though the serial ETT improved the accuracy. Functional studies showed that the G357S mutation increased caffeine sensitivity and store overload-induced calcium release activity under conditions that mimic catecholaminergic stress.nnnCONCLUSIONnOur study supports the use of genetic testing to identify individuals at risk of SCD to undertake prophylactic interventions. We also show that the pathogenic mechanisms of p.G357S_RyR2 appear to depend on β-adrenergic stimulation.

A novel missense mutation, I890T, in the pore region of cardiac sodium channel causes Brugada syndrome

Brugada syndrome (BrS) is a life-threatening, inherited arrhythmogenic syndrome associated with autosomal dominant mutations in SCN5A, the gene encoding the cardiac Na+ channel alpha subunit (Nav1.5). The aim of this work was to characterize the functional alterations caused by a novel SCN5A mutation, I890T, and thus establish whether this mutation is associated with BrS. The mutation was identified by direct sequencing of SCN5A from the proband’s DNA. Wild-type (WT) or I890T Nav1.5 channels were heterologously expressed in human embryonic kidney cells. Sodium currents were studied using standard whole cell patch-clamp protocols and immunodetection experiments were performed using an antibody against human Nav1.5 channel. A marked decrease in current density was observed in cells expressing the I890T channel (from −52.0±6.5 pA/pF, nu200a=u200a15 to −35.9±3.4 pA/pF, nu200a=u200a22, at −20 mV, WT and I890T, respectively). Moreover, a positive shift of the activation curve was identified (V 1/2u200a=u200a−32.0±0.3 mV, nu200a=u200a18, and −27.3±0.3 mV, nu200a=u200a22, WT and I890T, respectively). No changes between WT and I890T currents were observed in steady-state inactivation, time course of inactivation, slow inactivation or recovery from inactivation parameters. Cell surface protein biotinylation analyses confirmed that Nav1.5 channel membrane expression levels were similar in WT and I890T cells. In summary, our data reveal that the I890T mutation, located within the pore of Nav1.5, causes an evident loss-of-function of the channel. Thus, the BrS phenotype observed in the proband is most likely due to this mutation.

DiBAC4(3) hits a “sweet spot” for the activation of arterial large-conductance Ca2+-activated potassium channels independently of the β1-subunit

The voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC₄(3)] has been reported as a novel large-conductance Ca²⁺-activated K⁺ (BK) channel activator with selectivity for its β₁- or β₄-subunits. In arterial smooth muscle, BK channels are formed by a pore-forming α-subunit and a smooth muscle-abundant regulatory β₁-subunit. This tissue specificity has driven extensive pharmacological research aimed at regulating arterial tone. Using animals with a disruption of the gene for the β₁-subunit, we explored the effects of DiBAC₄(3) in native channels from arterial smooth muscle. We tested the hypothesis that, in native BK channels, activation by DiBAC₄(3) relies mostly on its α-subunit. We studied BK channels from wild-type and transgenic β₁-knockout mice in excised patches. BK channels from brain arteries, with or without the β₁-subunit, were similarly activated by DiBAC₄(3). In addition, we found that saturating concentrations of DiBAC₄(3) (~30 μM) promote an unprecedented persistent activation of the channel that negatively shifts its voltage dependence by as much as -300 mV. This sweet spot for persistent activation is independent of Ca²⁺ and/or the β₁₋₄-subunits and is fully achieved when DiBAC₄(3) is applied to the intracellular side of the channel. Arterial BK channel response to DiBAC₄(3) varies across species and/or vascular beds. DiBAC₄(3) unique effects can reveal details of BK channel gating mechanisms and help in the rational design of BK channel activators.