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Objectives Contrast-induced acute kidney injury (CI-AKI) may occur after percutaneous coronary intervention (PCI). Methods We evaluated patients with ST-elevation myocardial infarction (STEMI) undergoing emergency PCI with serial biomarkers. Results Of the 390 patients enrolled in the HORIZONS-AMI biomarker substudy, 56 (14.3%) developed AKI. In the AKI group, the levels of B-type natriuretic peptide were consistently higher than in the no-AKI group at baseline (P = 0.0327), hospital discharge (P = 0.0002), 30-day follow-up (P = 0.0193), and 1-year follow-up (P = 0.031). At hospital discharge, the AKI group had elevated biomarkers compared to the no-AKI group: D-dimer (P = 0.0066), C-reactive protein (P = 0.0468), endothelial cell-selective adhesion molecule (P = 0.0169), adiponectin (P = 0.0346), and von Willebrand factor (P = 0.0168); there was also a trend toward higher cystatin C (P = 0.0585) in the AKI group. Similar correlations between biomarker panel increase and the development of CI-AKI were consistent at baseline, 30-day, and 1-year follow-up. Chemokine (C-C motif) ligand 23 showed an opposite pattern with an increase at all time points in the no-AKI compared to the AKI group. Conclusions The risk of CI-AKI after primary PCI for STEMI may be associated with hemostatic imbalances, activation of procoagulants, decreased endogenous anticoagulants, enhanced inflammation, platelet activation, or decreased fibrinolytic activity. © 2014 Wiley Periodicals, Inc.

Objectives Our aim was to study the impact of delay from symptom onset to first coronary device on infarct size and clinical outcomes at 30 days and 1 year in patients with ST-segment elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention. Background Longer delay from symptom onset to reperfusion has been linked to increased mortality and worse clinical outcome. The mechanisms underpinning this association are not entirely clear. Methods The INFUSE-AMI trial (INFUSE-Anterior Myocardial Infarction) randomized patients with anterior STEMI undergoing primary percutaneous coronary intervention with bivalirudin anticoagulation within 5 h of symptom onset to intralesion (IL) bolus abciximab versus no abciximab and to thrombus aspiration versus no aspiration. The primary endpoint was contrast magnetic resonance infarct size (IS) (percentage of left ventricular mass) at 30 days. Time to reperfusion was classified as Results There were 280 patients (62%) with Conclusions In patients with large anterior myocardial infarction undergoing relatively early reperfusion, longer delays to reperfusion were associated with larger IS and 1-year mortality, but not with reduced reperfusion success. (The INFUSE - Anterior Myocardial Infarction [AMI] Study; NCT00976521 )

Both ST-segment resolution (STR) and myocardial blush grade (MBG) have prognostic utility after primary percutaneous coronary intervention (PCI) for ST-elevation myocardial infarction. We sought to clarify how frequently MBG and STR provide discordant measures of reperfusion success and to determine the independent prognostic significance of each on long-term outcomes.In HORIZONS-AMI, core laboratory measures of both MBG and STR were assessed in 2,367 patients undergoing primary PCI. Four groups were identified based on MBG (grades 2/3 vs 0/1) and STR (≥50% vs50%). A multivariable model identified predictors of death and major adverse cardiac events at 3 years.Myocardial blush grade 2/3 was achieved in 77.7% of patients, and STR ≥50% was achieved in 75.1% of patients. Myocardial blush grade and STR were discordant in 765 patients (30.9%). By multivariable analysis, MBG 2/3 compared with 0/1 was an independent predictor of lower mortality at 3 years (4.4% vs 8.4%, adjusted hazard ratio [HR] = 0.57 [0.39, 0.82], P = .003). In contrast, STR ≥50% compared with50% was not associated with mortality (5.1% vs 5.9%, adjusted HR = 1.11 [0.68, 1.56], P = .89). However, repeated revascularization at 3 years was less frequent when STR ≥50% (12.4% vs 17.6%, adjusted HR = 0.74 [0.58, 0.95], P = .02). In contrast, MBG 2/3 vs 0/1 was not associated with reduced repeated revascularization (13.6% vs 14.1%, adjusted HR = 1.02 [0.79, 1.33], P = .85).In HORIZONS-AMI, MBG and STR after primary PCI were concordant in only 70% of patients and provided complementary prognostic information. Myocardial blush grade predicted long-term survival, whereas STR predicted freedom from repeated revascularization.

Routine scheduled angiographic follow-up (SAF) after percutaneous coronary intervention (PCI) has been associated with a higher rate of target vessel revascularization (TVR). Its benefits are not known. SAF at 13 months after ST-segment elevation myocardial infarction (STEMI) was planned in the first 1,800 successfully stented patients enrolled in the Harmonizing Outcomes with RevascularIZatiON and Stents in Acute Myocardial Infarction (HORIZONS-AMI) trial. We compared the outcomes of patients with and without SAF at 1 year (before SAF) and at 3 years (after SAF). There were 1,197 patients (66.5% of expected) with and 2,207 patients without SAF. Prior to SAF, the 1-year composite rate of death or myocardial infarction (MI) was not significantly different between the 2 groups (2.7% vs. 3.9%, respectively, P = 0.06), although the rate of death was lower (0.1% vs. 2.2%, P

Background L-type calcium channel (LTCC) mutations have been associated with Brugada syndrome (BrS), short QT (SQT) syndrome, and Timothy syndrome (LQT8). Little is known about the extent to which LTCC mutations contribute to the J-wave syndromes associated with sudden cardiac death. Objective The purpose of this study was to identify mutations in the α1, β2, and α2δ subunits of LTCC (Ca v 1.2) among 205 probands diagnosed with BrS, idiopathic ventricular fibrillation (IVF), and early repolarization syndrome (ERS). CACNA1C, CACNB2b , and CACNA2D1 genes of 162 probands with BrS and BrS+SQT, 19 with IVF, and 24 with ERS were screened by direct sequencing. Methods/Results Overall, 23 distinct mutations were identified. A total of 12.3%, 5.2%, and 16% of BrS/BrS+SQT, IVF, and ERS probands displayed mutations in α1, β2, and α2δ subunits of LTCC, respectively. When rare polymorphisms were included, the yield increased to 17.9%, 21%, and 29.1% for BrS/BrS+SQT, IVF, and ERS probands, respectively. Functional expression of two CACNA1C mutations associated with BrS and BrS+SQT led to loss of function in calcium channel current. BrS probands displaying a normal QTc had additional variations known to prolong the QT interval. Conclusion The study results indicate that mutations in the LTCCs are detected in a high percentage of probands with J-wave syndromes associated with inherited cardiac arrhythmias, suggesting that genetic screening of Ca v genes may be a valuable diagnostic tool in identifying individuals at risk. These results are the first to identify CACNA2D1 as a novel BrS susceptibility gene and CACNA1C, CACNB2 , and CACNA2D1 as possible novel ERS susceptibility genes.

Background— Brugada syndrome, characterized by ST-segment elevation in the right precordial ECG leads and the development of life-threatening ventricular arrhythmias, has been associated with mutations in 6 different genes. We identify and characterize a mutation in a new gene. Methods and Results— A 64-year-old white male displayed a type 1 ST-segment elevation in V1 and V2 during procainamide challenge. Polymerase chain reaction-based direct sequencing was performed using a candidate gene approach. A missense mutation (L10P) was detected in exon 1 of SCN3B , the β3 subunit of the cardiac sodium channel, but not in any other gene known to be associated with Brugada syndrome or in 296 controls. Wild-type (WT) and mutant genes were expressed in TSA201 cells and studied using whole-cell patch-clamp techniques. Coexpression of SCN5A /WT+ SCN1B /WT+ SCN3B /L10P resulted in an 82.6% decrease in peak sodium current density, accelerated inactivation, slowed reactivation, and a −9.6-mV shift of half-inactivation voltage compared with SCN5A /WT+ SCN1B /WT+ SCN3B /WT. Confocal microscopy revealed that SCN5A /WT channels tagged with green fluorescent protein are localized to the cell surface when coexpressed with WT SCN1B and SCN3B but remain trapped in intracellular organelles when coexpressed with SCN1B /WT and SCN3B /L10P. Western blot analysis confirmed the presence of Na V β3 in human ventricular myocardium. Conclusions— Our results provide support for the hypothesis that mutations in SCN3B can lead to loss of transport and functional expression of the hNa v 1.5 protein, leading to reduction in sodium channel current and clinical manifestation of a Brugada phenotype.

Background— The Brugada syndrome, an inherited syndrome associated with a high incidence of sudden cardiac arrest, has been linked to mutations in 4 different genes, leading to a loss of function in sodium and calcium channel activity. Although the transient outward current ( I to ) is thought to play a prominent role in the expression of the syndrome, mutations in I to -related genes have not been identified as yet. Methods and Results— One hundred five probands with the Brugada syndrome were screened for ion channel gene mutations using single-strand conformation polymorphism electrophoresis and direct sequencing. A missense mutation (R99H) in KCNE3 ( MiRP2 ) was detected in 1 proband. The R99H mutation was found 4/4 phenotype-positive and 0/3 phenotype-negative family members. Chinese hamster ovary-K1 cells were cotransfected using wild-type (WT) or mutant KCNE3 and either WT KCND3 or KCNQ1. Whole-cell patch clamp studies were performed after 48 hours. Interactions between Kv4.3 and KCNE3 were analyzed in coimmunoprecipitation experiments in human atrial samples. Cotransfection of R99H- KCNE3 with KCNQ1 produced no alteration in tail current magnitude or kinetics. However, cotransfection of R99H KCNE3 with KCND3 resulted in a significant increase in the I to intensity compared with WT KCNE3 +KCND3. Using tissues isolated from the left atrial appendages of human hearts, we also demonstrate that K v 4.3 and KCNE3 can be coimmunoprecipitated. Conclusions— These results provide definitive evidence for a functional role of KCNE3 in the modulation of I to in the human heart and suggest that mutations in KCNE3 can underlie the development of the Brugada syndrome.

Background Atrial fibrillation (AF) is the most common clinical arrhythmia and a major cause of cardiovascular morbidity and mortality. Among the gene defects previously associated with AF is a gain of function of the slowly activating delayed rectifier potassium current IKs, secondary to mutations in KCNQ1. Coexpression of KCNE5, the gene encoding the MiRP4 β-subunit, has been shown to reduce IKs. Objective The purpose of this study was to test the hypothesis that mutations in KCNE5 are associated with AF in a large cohort of patients with AF. Methods One-hundred fifty-eight patients with AF were screened for mutations in the coding region of KCNE5. Results A missense mutation involving substitution of a phenylalanine for leucine at position 65 (L65F) was identified in one patient. This patient did not have a history of familial AF, and neither KCNQ1 nor KCNE2 mutations were found. Transient transfection of Chinese hamster ovary (CHO) cells expressing IKs(KCNQ1+KCNE1) with KCNE5 suppressed the developing and tail currents of IKs in a concentration-dependent manner. Transient transfection with KCNE5-L65F failed to suppress IKs, yielding a current indistinguishable from that recorded in the absence of KCNE5. Developing currents recorded during a test pulse to +60 mV and tail currents recorded upon repolarization to −40 mV both showed a significant concentration-dependent gain of function in IKs with expression of KCNE5-L65F vs KCNE5-WT. Conclusion The results of this study suggest that a missense mutation in KCNE5 may be associated with nonfamilial or acquired forms of AF. The arrhythmogenic mechanism most likely is a gain of function of IKs.

Introduction Long QT Syndrome (LQTS) is an inherited disorder characterized by prolonged QT intervals and life-threatening polymorphic ventricular tachyarrhythmias. LQT1 caused by KCNQ1 mutations is the most common form of LQTS. Methods and results Patients diagnosed with LQTS were screened for disease-associated mutations in KCNQ1, KCNH2, KCNE1, KCNE2, KCNJ2, and SCN5A. A novel mutation was identified in KCNQ1 caused by a three-base deletion at the position 824-826, predicting a deletion of phenylalanine at codon 275 in segment 5 of KCNQ1 (DeltaF275). Wild-type (WT) and DeltaF275-KCNQ1 constructs were generated and transiently transfected together with a KCNE1 construct in CHO-K1 cells to characterize the properties of the slowly activating delayed rectifier current (IKs) using conventional whole-cell patch-clamp techniques. Cells transfected with WT-KCNQ1 and KCNE1 (1:1.3 molar ratio) produced slowly activating outward current with the characteristics of IKs. Tail current density measured at -40 mV following a two-second step to +60 mV was 381.3 +/- 62.6 pA/pF (n = 11). Cells transfected with DeltaF275-KCNQ1 and KCNE1 exhibited essentially no current. (Tail current density: 0.8 +/- 2.1 pA/pF, n = 11, P = 0.00001 vs WT). Cotransfection of WT- and DeltaF275- KCNQ1 (50/50), along with KCNE1, produced little to no current (tail current density: 10.3 +/- 3.5 pA/pF, n = 11, P = 0.00001 vs WT alone), suggesting a potent dominant negative effect. Immunohistochemistry showed normal membrane trafficking of DeltaF275-KCNQ1. Conclusion Our data suggest that a DeltaF275 mutation in KCNQ1 is associated with a very potent dominant negative effect leading to an almost complete loss of function of IKs and that this defect underlies a LQT1 form of LQTS.

Background Ventricular tachycardia (VT) and ventricular fibrillation (VF) complicating Brugada syndrome, a genetic disorder linked to SCN5A mutations, and VF complicating acute myocardial infarction (AMI) both have been linked to phase 2 reentry. Objective Given the mechanistic similarities in arrhythmogenesis, the purpose of this study was to examine the contribution of SCN5A mutations to VT/VF complicating AMI. Methods Nineteen consecutive patients developing VF during AMI were enrolled in the study. Wild-type (WT) and mutant SCN5A genes were coexpressed with SCN1B in TSA201 cells and studied using whole-cell patch clamp techniques. Results Among the cohort of 19 patients, one missense mutation (G400A) in SCN5A was detected in a conserved region. An H558R polymorphism was detected on the same allele. Unlike the other 18 patients, who each developed 1–2 VF episodes during AMI, the mutation carrier developed six episodes of VT/VF within the first 12 hours. All VT/VF episodes were associated with ST-segment changes and were initiated by short-coupled extrasystoles. Flecainide and adenosine challenge performed to unmask Brugada and long QT syndromes both were negative. Peak G400A and G400A+H558R current were 70.7% and 88.4% less than WT current at −35 mV ( P ≤.001). G400A current decay was accelerated and steady-state inactivation was shifted −6.39 mV (V 1/2 = −98.9 ± 0.1 mV vs −92.5 ± 0.1 mV, P ≤.001). No mutations were detected in KCNH2, KCNQ1, KCNE1, or KCNE2 in the G400A patient. Conclusion We describe the first sodium channel mutation to be associated with the development of an arrhythmic storm during acute ischemia. These findings suggest that a loss of function in SCN5A may predispose to ischemia-induced arrhythmic storm.

Background The autonomic nervous system has been implicated in several arrhythmogenic diseases, including long QT syndrome type 3 (LQT3) and Brugada syndrome. Scarce information on the cellular components of the intrinsic cardiac ganglia from higher mammals has limited our understanding of the role of the autonomic nervous system in such diseases. Objectives The purpose of this study was to isolate and characterize the electrophysiologic properties of canine intracardiac neurons. Methods Action potentials (APs) and ionic currents were studied in enzymatically dissociated canine intracardiac neurons under current and voltage clamp conditions. Immunohistochemical and reverse transcription-polymerase chain reaction analysis was performed using freshly isolated intracardiac ganglia. Results APs recorded from intracardiac neurons displayed a tetrodotoxin-resistant (TTX-R) component. TTX-R APs were abolished in the absence of sodium but persisted in the absence of external calcium. Immunohistochemical studies showed the presence of TTX-R sodium channels in these ganglia. Sodium currents were characterized by two components with different affinities for TTX: a tetrodotoxin-sensitive (TTX-S) component and a TTX-R component. TTX-S current inactivation was characteristic of neuronal sodium currents, whereas TTX-R current inactivation time constants were similar to those previously reported for Na v 1.5 channels. TTX sensitivity (IC 50 = 1.17 μM) of the TTX-R component was in the range reported for Na v 1.5 channels. Expression of Na v 1.5 channels in intracardiac ganglia was confirmed by PCR analysis and sequencing. Conclusion Our results suggest that canine intracardiac neurons functionally express Na v 1.5 channels. These findings open an exciting new door to our understanding of autonomically modulated arrhythmogenic diseases linked to mutations in Na v 1.5 channels, including Brugada syndrome and LQT3.

Rapid reperfusion with primary percutaneous coronary intervention improves survival in patients with ST-segment elevation myocardial infarction. Preprocedural cardiopulmonary instability and adverse events (IAE) may delay reperfusion time and worsen prognosis. The aim of this study was to evaluate the relation between preprocedural cardiopulmonary IAE, door-to-balloon time (DBT), and outcomes in the Harmonizing Outcomes With Revascularization and Stents in AMI (HORIZONS-AMI) trial. Preprocedural cardiopulmonary IAE included sustained ventricular or supraventricular tachycardia or fibrillation requiring cardioversion or defibrillation, heart block or bradycardia requiring pacemaker implantation, severe hypotension requiring vasopressors or intra-aortic balloon counterpulsation, respiratory failure requiring mechanical ventilation, and cardiopulmonary resuscitation. Three-year outcomes of patients with and without IAE according to DBT were compared. Among 3,602 patients, 159 (4.4%) had ≥1 IAE. DBT did not differ significantly in patients with and without IAE; however, patients with IAE were less likely to have Thrombolysis In Myocardial Infarction (TIMI) grade 3 flow after percutaneous coronary intervention. Mortality at 3 years was significantly higher in patients with versus those without IAE (17.0% vs 6.3%, p0.0001), and IAE was an independent predictor of mortality, whereas DBT was not. However, a significant interaction was present such that 3-year mortality was reduced in patients with DBT99 minutes (the median) versus ≥99 minutes to a greater extent in patients with IAE (9.9% vs 20.7%, hazard ratio 0.43, 95% confidence interval 0.16 to 1.16) compared with those without IAE (5.0% vs 7.2%, hazard ratio 0.69, 95% confidence interval 0.50 to 0.95) (p for interaction=0.004). In conclusion, IAE before PCI is an independent predictor of death and identifies a high-risk group in whom faster reperfusion may be particularly important to improve survival.

Our aim was to study the impact of delay from symptom onset to first coronary device on infarct size and clinical outcomes at 30 days and 1 year in patients with ST-segment elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention.Longer delay from symptom onset to reperfusion has been linked to increased mortality and worse clinical outcome. The mechanisms underpinning this association are not entirely clear.The INFUSE-AMI trial (INFUSE-Anterior Myocardial Infarction) randomized patients with anterior STEMI undergoing primary percutaneous coronary intervention with bivalirudin anticoagulation within 5 h of symptom onset to intralesion (IL) bolus abciximab versus no abciximab and to thrombus aspiration versus no aspiration. The primary endpoint was contrast magnetic resonance infarct size (IS) (percentage of left ventricular mass) at 30 days. Time to reperfusion was classified as3 versus ≥3 h.There were 280 patients (62%) with3-h delay and 170 patients (38%) with ≥3-h delay. Patients with longer delay were significantly older, more often women, and diabetic. Earlier reperfusion was not associated with higher rates of final Thrombolysis In Myocardial Infarction flow grade 3 or myocardial blush grade 2/3, but was an independent predictor of smaller IS (p = 0.02 by multivariable linear regression). Mortality at 1 year was reduced in patients with shorter delay to reperfusion (4.0% vs. 9.2%, p = 0.02).In patients with large anterior myocardial infarction undergoing relatively early reperfusion, longer delays to reperfusion were associated with larger IS and 1-year mortality, but not with reduced reperfusion success. (The INFUSE - Anterior Myocardial Infarction [AMI] Study; NCT00976521).

Routine scheduled angiographic follow-up (SAF) after percutaneous coronary intervention (PCI) has been associated with a higher rate of target vessel revascularization (TVR). Its benefits are not known. SAF at 13 months after ST-segment elevation myocardial infarction (STEMI) was planned in the first 1,800 successfully stented patients enrolled in the Harmonizing Outcomes with RevascularIZatiON and Stents in Acute Myocardial Infarction (HORIZONS-AMI) trial. We compared the outcomes of patients with and without SAF at 1 year (before SAF) and at 3 years (after SAF). There were 1,197 patients (66.5% of expected) with and 2,207 patients without SAF. Prior to SAF, the 1-year composite rate of death or myocardial infarction (MI) was not significantly different between the 2 groups (2.7% vs. 3.9%, respectively, P=0.06), although the rate of death was lower (0.1% vs. 2.2%, P0.0001), nor were there differences in the 1-year rates of TVR, stent thrombosis or major adverse cardiac and cerebral events). At 3 years, death or MI rates were again similar between the groups (8.3% vs. 9.5%, P=0.22), but TVR was more common in the SAF group (17.0% vs. 8.6%, P0.0001), due to an increase in TVR at time of SAF. In the SAF group, patients in whom TVR was performed before or after the 13-month SAF window had markedly higher 3-year rates of MI and stent thrombosis than patients in whom TVR was performed during SAF or not at all. In conclusion, SAF after primary PCI in STEMI is associated with doubling of the rate of revascularization without an improvement in death or MI, and therefore cannot be recommended.

Background Although QT prolongation following myocardial infarction (MI) is generally moderate, cases with marked QT prolongation leading to life-threatening torsades de pointes (TdP) have been described. Objective To investigate the genetic substrate of this phenomenon. Methods We studied 13 patients who developed TdP in the subacute phase of MI (2–11 days) and a group of 133 ethnically matched controls with uncomplicated MI. Long QT syndrome genes and the KCNH2 -K897T polymorphism were screened by using denaturing high-performance liquid chromatography plus direct sequencing and a specific TaqMan assay, respectively. Results Two of the 13 patients (15%) who presented with QT prolongation and TdP were found to carry long QT syndrome mutations ( KCNH2 -R744X and SCN5A -E446K). Nine of the remaining 11 patients (82%) carried the KCNH2- K897T polymorphism, which was present in 35% of the controls ( P = .0035). Thus, patients with an acute MI carrying the KCNH2 -K897T polymorphism had an 8-fold greater risk of experiencing TdP compared with controls (95% confidence interval=2–40). Conclusions Our data suggest that the common K897T polymorphism is associated with an increased risk of TdP developing in the subacute phase of MI. Our findings support the concept that the electrical remodeling associated with this healing phase of MI may unmask a genetic substrate predisposing to a time-limited development of life-threatening arrhythmias. They also provide the first line of evidence in support of the hypothesis that a common polymorphism, previously described as a modifier of the severity of LQTS, may increase the risk of life-threatening arrhythmias in a much more prevalent cardiac disease such as myocardial infarction.

Coronary heart disease and its main complication, myocardial infarction is leading cause of death worldwide. Over the past years, much progress has been made in the pharmacotherapy of major risk factors like dyslipidemias, diabetes mellitus and hypertension. The targeting of coronary risk factors coupled with advances in the management of coronary artery disease has improved patient survival. However, the incidence of cardiovascular disease is projected to continue to rise and the identification of individuals at risk should improve beyond the traditional models of global risk factor scoring. In the past few years, important progresses have been made in the area of genomics, especially with the completion of the human genome-sequencing Consortium of 2004, proteomics and imaging. This progress will promote a better understanding of cardiovascular risk assessments and disease prediction, thus allowing earlier preventive strategies to prevent and improve cardiovascular outcomes. These genomic advances have improved characterization of disease pathology especially at the molecular level with the discovery and introduction of genetic markers, single nucleotide polymorphisms (SNPs), and haplotype blocks.

Background Brugada syndrome (BrS) is a common heritable channelopathy. Mutations in the SCN5A -encoded sodium channel (BrS1) culminate in the most common genotype. Objective This study sought to perform a retrospective analysis of BrS databases from 9 centers that have each genotyped >100 unrelated cases of suspected BrS. Methods Mutational analysis of all 27 translated exons in SCN5A was performed. Mutation frequency, type, and localization were compared among cases and 1,300 ostensibly healthy volunteers including 649 white subjects and 651 nonwhite subjects (blacks, Asians, Hispanics, and others) that were genotyped previously. Results A total of 2,111 unrelated patients (78% male, mean age 39 ± 15 years) were referred for BrS genetic testing. Rare mutations/variants were more common among BrS cases than control subjects (438/2,111, 21% vs. 11/649, 1.7% white subjects and 31/651, 4.8% nonwhite subjects, respectively, P −53 ). The yield of BrS1 genetic testing ranged from 11% to 28% ( P = .0017). Overall, 293 distinct mutations were identified in SCN5A: 193 missense, 32 nonsense, 38 frameshift, 21 splice-site, and 9 in-frame deletions/insertions. The 4 most frequent BrS1-associated mutations were E1784K (14×), F861WfsX90 (11×), D356N (8×), and G1408R (7×). Most mutations localized to the transmembrane-spanning regions. Conclusion This international consortium of BrS genetic testing centers has added 200 new BrS1-associated mutations to the public domain. Overall, 21% of BrS probands have mutations in SCN5A compared to the 2% to 5% background rate of rare variants reported in healthy control subjects. Additional studies drawing on the data presented here may help further distinguish pathogenic mutations from similarly rare but otherwise innocuous ones found in cases.

Recent reports have highlighted the importance of a family history of sudden death as a risk for ventricular fibrillation in patients experiencing an acute myocardial infarction (AMI), pointing to the possibility of a genetic predisposition. This report briefly reviews two recent studies designed to examine the hypothesis that there is a genetic predisposition to the development of arrhythmias associated with AMI. Ventricular tachycardia and fibrillation (VT/VF) complicating AMI as well as the arrhythmias associated with Brugada syndrome, a genetic disorder linked to SCN5A mutations, have both been linked to phase 2 reentry. Because of these mechanistic similarities in arrhythmogenesis, we examined the contribution of SCN5A mutations to VT/VF complicating AMI in patients developing VF during AMI. A missense mutation in SCN5A was found in a patient who developed an arrhythmic electrical storm during an evolving MI. All VT/VF episodes were associated with ST segment changes and were initiated by short-coupled extrasystoles. The G400A mutation and a H558R polymorphism were on the same allele and functional expression in TSA201 demonstrated a loss of function of sodium channel activity. These results suggest that a subclinical mutation in SCN5A resulting in a loss of function may predispose to life-threatening arrhythmias during acute ischemia. In another cohort of patients who developed long QT intervals and Torsade de Pointes (TdP) arrhythmias in days 2–11 following an AMI, a genetic screen of all long QT genes was performed. Six of eight patients (75%) in this group displayed the same polymorphism in KCNH2, which encodes the α subunit of the rapidly activating delayed rectifier potassium current, IKr. The K897T polymorphism was detected in only 3 of 14 patients with uncomplicated myocardial infarction (MI) and has been detected in 33% of the Caucasian population. Expression of this polymorphism has previously been shown to cause a loss of function in HERG current consistent with the long QT phenotype. These observations suggest a genetic predisposition to the development of long QT intervals and TdP in the days following an AMI. These preliminary studies provide support for the hypothesis that there is a genetic predisposition to the type and severity of arrhythmias that develop during and after an acute myocardial infarction and that additional studies are warranted.

Background— Cardiac ion channelopathies are responsible for an ever-increasing number and diversity of familial cardiac arrhythmia syndromes. We describe a new clinical entity that consists of an ST-segment elevation in the right precordial ECG leads, a shorter-than-normal QT interval, and a history of sudden cardiac death. Methods and Results— Eighty-two consecutive probands with Brugada syndrome were screened for ion channel gene mutations with direct sequencing. Site-directed mutagenesis was performed, and CHO-K1 cells were cotransfected with cDNAs encoding wild-type or mutant CACNB2b (Ca vβ2b ), CACNA2D1 (Ca vα2δ1 ), and CACNA1C tagged with enhanced yellow fluorescent protein (Ca v 1.2). Whole-cell patch-clamp studies were performed after 48 to 72 hours. Three probands displaying ST-segment elevation and corrected QT intervals ≤360 ms had mutations in genes encoding the cardiac L-type calcium channel. Corrected QT ranged from 330 to 370 ms among probands and clinically affected family members. Rate adaptation of QT interval was reduced. Quinidine normalized the QT interval and prevented stimulation-induced ventricular tachycardia. Genetic and heterologous expression studies revealed loss-of-function missense mutations in CACNA1C (A39V and G490R) and CACNB2 (S481L) encoding the α 1 - and β 2b -subunits of the L-type calcium channel. Confocal microscopy revealed a defect in trafficking of A39V Ca v 1.2 channels but normal trafficking of channels containing G490R Ca v 1.2 or S481L Ca vβ2b -subunits. Conclusions— This is the first report of loss-of-function mutations in genes encoding the cardiac L-type calcium channel to be associated with a familial sudden cardiac death syndrome in which a Brugada syndrome phenotype is combined with shorter-than-normal QT intervals.

SummaryRhythmic cardiac contractions depend on the organized propagation of depolarizing and repolarizing wavefronts. Repolarization is spatially heterogeneous and depends largely on gradients of potassium currents. Gradient disruption in heart disease may underlie susceptibility to fatal arrhythmias, but it is not known how this gradient is established. We show that, in mice lacking the homeodomain transcription factor Irx5, the cardiac repolarization gradient is abolished due to increased Kv4.2 potassium-channel expression in endocardial myocardium, resulting in a selective increase of the major cardiac repolarization current, Ito,f, and increased susceptibility to arrhythmias. Myocardial Irx5 is expressed in a gradient opposite that of Kv4.2, and Irx5 represses Kv4.2 expression by recruiting mBop, a cardiac transcriptional repressor. Thus, an Irx5 repressor gradient negatively regulates potassium-channel-gene expression in the heart, forming an inverse Ito,f gradient that ensures coordinated cardiac repolarization while also preventing arrhythmias.

The Brugada syndrome (BS) is characterized by ST segment elevation in the right precordial leads and sudden cardiac death. The disease is linked to mutations in SCN5A in approximately 20% of cases. We collected a large family with BS and have identified a novel intronic mutation. We performed the clinical, genetic, molecular and biophysical characterization of this disease-causing mutation. With direct sequencing we identified an intronic insertion of TGGG 5 bp from the end of the Exon 27 of SCN5A. For transcript analysis, we investigated Epstein-Barr-transformed lymphoblastoid cell lines from patients and controls. Total RNA was extracted and RT-PCR experiments were performed to analyze the splicing patterns in exon 27 and 28. We identified two bands, one of the expected size and the other which showed a 96 bp deletion in exon 27, leading to a 32 amino acid in-frame deletion involving segments 2 and 3 of Domain IV of the SCN5A protein. This finding indicates that the intronic mutation creates a cryptic splice site inside Exon 27. Biophysical analysis using whole-cell patch-clamp techniques showed a complete loss of function of the mutated channels when heterologously expressed. In summary, this is the first report of a dysfunctional sodium channel created by an intronic mutation giving rise to cryptic splice site activation in SCN5A in a family with the BS. The deletion of fragments of segments 2 and 3 of Domain IV leads to complete loss of function, consistent with the biophysical data found in several mutations causing BS.

Background— The Brugada syndrome is an arrhythmogenic disease caused in part by mutations in the cardiac sodium channel gene, SCN5A. The electrocardiographic pattern characteristic of the syndrome is dynamic and is often absent in affected individuals. Sodium channel blockers are effective in unmasking carriers of the disease. However, the value of the test remains controversial. Methods and Results— We studied 147 individuals representing 4 large families with SCN5A mutations. Of these, 104 were determined to be at possible risk for Brugada syndrome and underwent both electrocardiographic and genetic evaluation. Twenty-four individuals displayed an ECG diagnostic of Brugada syndrome at baseline. Of the remaining, 71 received intravenous ajmaline. Of the 35 genetic carriers who received ajmaline, 28 had a positive test and 7 a negative ajmaline test. The sensitivity, specificity, and positive and negative predictive values of the drug challenge were 80% (28:35), 94.4% (34:36), 93.3% (28:30), and 82.9% (34:41), respectively. Penetrance of the disease phenotype increased from 32.7% to 78.6% with the use of sodium channel blockers. In the absence of ST-segment elevation under baseline conditions, a prolonged P-R interval, but not incomplete right bundle-branch block or early repolarization patterns, indicates a high probability of an SCN5A mutation carrier. Conclusions— In families with Brugada syndrome, the data suggest that ajmaline testing is valuable in the diagnosis of SCN5A carriers. In the absence of ST-segment elevation at baseline, family members with first-degree atrioventricular block should be suspected of carrying the mutation. An ajmaline test is often the key to making the proper diagnosis in these patients.

Background— Sudden cardiac death takes the lives of more than 300 000 Americans annually. Malignant ventricular arrhythmias occurring in individuals with structurally normal hearts account for a subgroup of these sudden deaths. The present study describes the genetic basis for a new clinical entity characterized by sudden death and short-QT intervals in the ECG. Methods and Results— Three families with hereditary short-QT syndrome and a high incidence of ventricular arrhythmias and sudden cardiac death were studied. In 2 of them, we identified 2 different missense mutations resulting in the same amino acid change (N588K) in the S5-P loop region of the cardiac I Kr channel HERG (KCNH2). The mutations dramatically increase I Kr , leading to heterogeneous abbreviation of action potential duration and refractoriness, and reduce the affinity of the channels to I Kr blockers. Conclusions— We demonstrate a novel genetic and biophysical mechanism responsible for sudden death in infants, children, and young adults caused by mutations in KCNH2. The occurrence of sudden cardiac death in the first 12 months of life in 2 patients suggests the possibility of a link between KCNH2 gain of function mutations and sudden infant death syndrome. KCNH2 is the binding target for a wide spectrum of cardiac and noncardiac pharmacological compounds. Our findings may provide better understanding of drug interaction with KCNH2 and have implications for diagnosis and therapy of this and other arrhythmogenic diseases.

We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin . Three IS 240 -like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis . Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti , Anopheles stephensi , and Culex quinquefasciatus , including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus .

In the same way that cry genes, coding for larvicidal delta endotoxins, constitute a large and diverse gene family, the cyt genes for hemolytic toxins seem to compose another set of highly related genes in Bacillus thuringiensis . Although the occurrence of Cyt hemolytic factors in B. thuringiensis has been typically associated with mosquitocidal strains, we have recently shown that cyt genes are also present in strains with different pathotypes; this is the case for the morrisoni subspecies, which includes strains biologically active against dipteran, lepidopteran, and coleopteran larvae. In addition, while one Cyt type of protein has been described in all of the mosquitocidal strains studied so far, the present study confirms that at least two Cyt toxins coexist in the more toxic antidipteran strains, such as B. thuringiensis subsp. israelensis and subsp. morrisoni PG14, and that this could also be the case for many others. In fact, PCR screening and Western blot analysis of 50 B. thuringiensis strains revealed that cyt 2-related genes are present in all strains with known antidipteran activity, as well as in some others with different or unknown host ranges. Partial DNA sequences for several of these genes were determined, and protein sequence alignments revealed a high degree of conservation of the structural domains. These findings point to an important biological role for Cyt toxins in the final in vivo toxic activity of many B. thuringiensis strains.

The presence of the Φ105 cts 23 mutant prophage in Bacillus subtilis induces a series of pleiotropic effects that could be ascribed to an anti-SOS activity. In order to circumvent the phage function responsible for this phenomenon, the cts 23 mutant repressor was cloned and sequenced. The isolated repressor reduced the survival capacity of the host cells after mitomycin C or nalidixic acid treatments and lowered the spontaneous reversion frequency. When SOS induction kinetics were studied, low or null induction of the damage-inducible din 22:: Lac Z fusion was observed. In contrast, the presence of the wild-type prophage amplified the SOS response. Sequencing of the mutant repressor revealed that the cts 23 mutation is a T→C transition affecting the 5′ closest codon to one of the two reported DNA binding domains.

Introduction: Inherited loss of function mutations in SCN5A, the gene that encodes the α-subunit of the human cardiac sodium channel (hNav1.5), have been linked to overlapping syndromes including cardiac conduction disease (CCD) and Brugada syndrome (BrS). The mechanisms responsible for the development of one without the other are poorly understood.Methods: Direct sequencing analysis was performed in a family with CCD. Wild type (WT) and variant channels were co-expressed with CD8 cDNA in TSA201 cells for electrophysiological study. Green fluorescent protein (GFP)-fused WT or mutant SCN5A genes were used for confocal microscopy to assess channel trafficking.Results: A novel SCN5A missense mutation, P1008S, was identified in all family members displaying 1st degree AV block, but not in unaffected family members nor in 430 reference alleles. Peak P1008S current was 11.77% of WT (p