Barry London

Mutation in Glycerol-3-Phosphate Dehydrogenase 1–Like Gene (GPD1-L) Decreases Cardiac Na+ Current and Causes Inherited Arrhythmias

Background— Brugada syndrome is a rare, autosomal-dominant, male-predominant form of idiopathic ventricular fibrillation characterized by a right bundle-branch block and ST elevation in the right precordial leads of the surface ECG. Mutations in the cardiac Na+ channel SCN5A on chromosome 3p21 cause ≈20% of the cases of Brugada syndrome; most mutations decrease inward Na+ current, some by preventing trafficking of the channels to the surface membrane. We previously used positional cloning to identify a new locus on chromosome 3p24 in a large family with Brugada syndrome and excluded SCN5A as a candidate gene. Methods and Results— We used direct sequencing to identify a mutation (A280V) in a conserved amino acid of the glycerol-3-phosphate dehydrogenase 1–like (GPD1-L) gene. The mutation was present in all affected individuals and absent in >500 control subjects. GPD1-L RNA and protein are abundant in the heart. Compared with wild-type GPD1-L, coexpression of A280V GPD1-L with SCN5A in HEK cells reduced inward Na+ currents by ≈50% (P<0.005). Wild-type GPD1-L localized near the cell surface to a greater extent than A280V GPD1-L. Coexpression of A280V GPD1-L with SCN5A reduced SCN5A cell surface expression by 31±5% (P=0.01). Conclusions— GPD1-L is a novel gene that may affect trafficking of the cardiac Na+ channel to the cell surface. A GPD1-L mutation decreases SCN5A surface membrane expression, reduces inward Na+ current, and causes Brugada syndrome.

Design of a Phase 1/2 Trial of Intracoronary Administration of AAV1/SERCA2a in Patients With Heart Failure

BACKGROUND Heart failure (HF) remains a major cause of morbidity and mortality in North America. With an aging population and an unmet clinical need by current pharmacologic and device-related therapeutic strategies, novel treatment options for HF are being explored. One such promising strategy is gene therapy to target underlying molecular anomalies in the dysfunctional cardiomyocyte. Prior animal and human studies have documented decreased expression of SERCA2a, a major cardiac calcium cycling protein, as a major defect found in HF. METHODS AND RESULTS We hypothesize that increasing the activity of SERCA2a in patients with moderate to severe HF will improve their cardiac function, disease status, and quality of life. Gene transfer of SERCA2a will be performed via an adeno-associated viral (AAV) vector, derived from a nonpathogenic virus with long-term transgene expression as well as a clinically established favorable safety profile. CONCLUSIONS We describe the design of a phase 1 clinical trial of antegrade epicardial coronary artery infusion (AECAI) administration of AAVI/SERCA2a (MYDICAR) to subjects with HF divided into 2 stages: in Stage 1, subjects will be assigned open-label MYDICAR in one of up to 4 sequential dose escalation cohorts; in Stage 2, subjects will be randomized in parallel to 2 or 3 doses of MYDICAR or placebo in a double-blinded manner.

Molecular basis of transient outward K+ current diversity in mouse ventricular myocytes.

1 Two kinetically and pharmacologically distinct transient outward K+ currents, referred to as Ito,f and Ito,s, have been distinguished in mouse left ventricular myocytes. Ito,f is present in all left ventricular apex cells and in most left ventricular septum cells, whereas Ito,s is identified exclusively in left ventricular septum cells. 2 Electrophysiological recordings from ventricular myocytes isolated from animals with a targeted deletion of the Kv1.4gene (Kv1.4−/− mice) reveal that Ito,s is undetectable in cells isolated from the left ventricular septum (n= 26). Ito,f density in both apex and septum cells, in contrast, is not affected by deletion of Kv1.4. 3 Neither the 4‐AP‐sensitive, slowly inactivating K+ current, IK,slow, nor the steady‐state non‐inactivating K+ current, ISS, is affected in Kv1.4−/− mouse left ventricular cells. 4 In myocytes isolated from transgenic mice expressing a dominant negative Kv4.2 α subunit, Kv4.2W362F, Ito,f is eliminated in both left ventricular apex and septum cells. In addition, a slowly inactivating transient outward K+ current similar to Ito,s in wild‐type septum cells is evident in myocytes isolated from left ventricular apex of Kv4.2W362F‐expressing transgenics. The density of Ito,s in septum cells, however, is unaffected by Kv4.2W362F expression. 5 Western blots of fractionated mouse ventricular membrane proteins reveal a significant increase in Kv1.4 protein level in Kv4.2W362F‐expressing transgenic mice. The protein levels of other Kv α subunits, Kv1.2 and Kv2.1, in contrast, are not affected by the expression of the Kv4.2W362F transgene. 6 The results presented here demonstrate that the molecular correlates of Ito,f and Ito,s in adult mouse ventricle are distinct. Kv1.4 underlies mouse ventricular septum Ito,s, whereas Kv α subunits of the Kv4 subfamily underlie mouse ventricular apex and septum Ito,f. The appearance of the slow transient outward K+ current in Kv4.2W362F‐expressing left ventricular apex cells with properties indistinguishable from Ito,s in wild‐type cells is accompanied by an increase in Kv1.4 protein expression, suggesting that the upregulation of Kv1.4 underlies the observed electrical remodeling in Kv4.2W362F‐expressing transgenics.

Enhanced Dispersion of Repolarization and Refractoriness in Transgenic Mouse Hearts Promotes Reentrant Ventricular Tachycardia

The heterogeneous distribution of ion channels in ventricular muscle gives rise to spatial variations in action potential (AP) duration (APD) and contributes to the repolarization sequence in healthy hearts. It has been proposed that enhanced dispersion of repolarization may underlie arrhythmias in diseases with markedly different causes. We engineered dominant negative transgenic mice that have prolonged QT intervals and arrhythmias due to the loss of a slowly inactivating K(+) current. Optical techniques are now applied to map APs and investigate the mechanisms underlying these arrhythmias. Hearts from transgenic and control mice were isolated, perfused, stained with di-4-ANEPPS, and paced at multiple sites to optically map APs, activation, and repolarization sequences at baseline and during arrhythmias. Transgenic hearts exhibited a 2-fold prolongation of APD, less shortening (8% versus 40%) of APDs with decreasing cycle length, altered restitution kinetics, and greater gradients of refractoriness from apex to base compared with control hearts. A premature impulse applied at the apex of transgenic hearts produced sustained reentrant ventricular tachycardia (n=14 of 15 hearts) that did not occur with stimulation at the base (n=8) or at any location in control hearts (n=12). In transgenic hearts, premature impulses initiated reentry by encountering functional lines of conduction block caused by enhanced dispersion of refractoriness. Reentrant VT had stable (>30 minutes) alternating long/short APDs associated with long/short cycle lengths and T wave alternans. Thus, optical mapping of genetically engineered mice may help elucidate some electrophysiological mechanisms that underlie arrhythmias and sudden death in human cardiac disorders.

Molecular and Functional Characterization of Novel Glycerol-3-Phosphate Dehydrogenase 1–Like Gene (GPD1-L) Mutations in Sudden Infant Death Syndrome

Background— Autopsy-negative sudden unexplained death, including sudden infant death syndrome, can be caused by cardiac channelopathies such as Brugada syndrome (BrS). Type 1 BrS, caused by mutations in the SCN5A-encoded sodium channel, accounts for ≈20% of BrS. Recently, a novel mutation in the glycerol-3-phosphate dehydrogenase 1–like gene (GPD1-L) disrupted trafficking of SCN5A in a multigenerational family with BrS. We hypothesized that mutations in GPD1-L may be responsible for some cases of sudden unexplained death/sudden infant death syndrome. Methods and Results— Using denaturing high-performance liquid chromatography and direct DNA sequencing, we performed comprehensive open-reading frame/splice site mutational analysis of GPD1-L on genomic DNA extracted from necropsy tissue of 83 unrelated cases of sudden unexplained death (26 females, 57 males; average age, 14.6±10.7 years; range, 1 month to 48 years). A putative, sudden unexplained death–associated GPD1-L missense mutation, E83K, was discovered in a 3-month-old white boy. Further mutational analysis was then performed on genomic DNA derived from a population-based cohort of 221 anonymous cases of sudden infant death syndrome (84 females, 137 males; average age, 3±2 months; range, 3 days to 12 months), revealing 2 additional mutations, I124V and R273C, in a 5-week-old white girl and a 1-month-old white boy, respectively. All mutations occurred in highly conserved residues and were absent in 600 reference alleles. Compared with wild-type GPD1-L, GPD1-L mutations coexpressed with SCN5A in heterologous HEK cells produced a significantly reduced sodium current (P<0.01). Adenovirus-mediated gene transfer of the E83K–GPD1-L mutation into neonatal mouse myocytes markedly attenuated the sodium current (P<0.01). These decreases in current density are consistent with sodium channel loss-of-function diseases like BrS. Conclusions— The present study is the first to report mutations in GPD1-L as a pathogenic cause for a small subset of sudden infant death syndrome via a secondary loss-of-function mechanism whereby perturbations in GPD1-L precipitate a marked decrease in the peak sodium current and a potentially lethal BrS-like proarrhythmic substrate.

Omega-3 Fatty Acids and Cardiac Arrhythmias: Prior Studies and Recommendations for Future Research A Report from the National Heart, Lung, and Blood Institute and Office of Dietary Supplements Omega-3 Fatty Acids and Their Role in Cardiac Arrhythmogenesis Workshop

Compared with prehistoric times, the ratio of n-6 to n-3 fatty acids in the modern diet has increased ≈10-fold to 20:1.1,2 A substantial body of evidence suggests that n-3 polyunsaturated fatty acids (PUFAs) provide cardiovascular protection and prevent arrhythmias.3–5 This has led to the recommendation by the American Heart Association that all adults eat fatty fish at least 2 times per week and that patients with coronary heart disease (CHD) are advised to consume ≈1 g/d of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) combined.6,7 The evidence base is not entirely consistent, and a number of randomized trials have failed to show a protective effect of n-3 PUFAs against arrhythmias.8–10 This has led to some uncertainty regarding the appropriate recommendations for their use.11 The present review originates from the Omega-3 Fatty Acids and Their Role in Cardiac Arrhythmogenesis Workshop sponsored by the National Heart, Lung, and Blood Institute and the Office of Dietary Supplements on August 29–30, 2005, and includes the findings from the recently published trials. Data from epidemiological studies, randomized clinical trials, animal studies, and basic science mechanistic studies on the role of n-3 PUFAs in arrhythmia prevention are examined. Areas in which the data are conflicting or our current knowledge is lacking are emphasized. Fatty acids are classified by the length of the carbon chain (long chain, n=20 to 22; intermediate chain, n=18) and the number of double bonds (saturated, monounsaturated, polyunsaturated).1,2 For PUFAs, the location of the first double bond relative to the -CH3 or omega (n-) end is given. Long- and intermediate-chain fatty acids must be ingested as part of the diet because they cannot be synthesized by humans and are therefore referred to as essential. The most common dietary fatty acids include (1) the omega-6 linoleic acid …

Impairment of hypoxic pulmonary vasoconstriction in mice lacking the voltage-gated potassium channel Kv1.5

Hypoxic pulmonary vasoconstriction (HPV) is initiated by the inhibition of several 4‐aminopyridine (4‐AP)‐sensitive, voltage‐gated, K+ channels (Kv). Several O2‐sensitive candidate channels (Kv1.2, Kv1.5, Kv2.1, and Kv3.1b) have been proposed, based on similarities between their characteristics in expression systems and the properties of the O2‐sensitive K+ current (IK) in pulmonary artery smooth muscle cells (PASMCs). We used gene targeting to delete Kv1.5 in mice by creating a SWAP mouse that is functionally a Kv1.5 knockout. We hypothesized that SWAP mice would display impaired HPV. The Kv1.5 α‐subunits present in the endothelium and PASMCs of wild‐type mice were absent in the lungs of SWAP mice, whereas expression of other channels Kv (1.1, 1.2, 2.1, 3.1, 4.3), Kir 3.1, Kir 6.1, and BKCa was unaltered. In isolated lungs and resistance PA rings, HPV was reduced significantly in SWAP versus wild‐type mice. Consistent with this finding, PASMCs from SWAP PAs were slightly depolarized and lacked IKv1.5, a 4‐AP and hypoxia‐sensitive component of IK that activated between ‐50 mV and ‐30 mV. We conclude that a K+ channel containing Kv1.5 α‐subunits is an important effector of HPV in mice.

Upsurge in T-Wave Alternans and Nonalternating Repolarization Instability Precedes Spontaneous Initiation of Ventricular Tachyarrhythmias in Humans

Background— Analysis of repolarization instability, manifested by T-wave alternans (TWA), has proved useful for arrhythmia risk assessment. However, temporal relations between TWA and the spontaneous initiation of ventricular tachyarrhythmias (VTA) in humans are unknown. We examined continuous dynamics of repolarization in Holter electrocardiograms with spontaneous sustained (>30 seconds) VTA. Methods and Results— Ambulatory electrocardiograms from 42 patients (79% with ischemic heart disease; left ventricular ejection fraction, 37±15%) were digitized, and the lead with the highest magnitude of the T wave was selected for analysis. TWA was examined by the modified moving average and intrabeat average analyses. To examine non-TWA (longer-period) oscillations in the repolarization segment, spectral energy of oscillations of consecutive T-wave amplitudes was calculated with the use of the short-time Fourier transform. Heart rate variability was assessed with the Fourier transform as well. TWA increased before the onset of VTA and reached a peak value of 23.6±11.7 &mgr;V 10 minutes before the event (P=0.0007). Spectral power of the oscillations of consecutive T-wave amplitudes increased nonuniformly, with the greatest increase in the respiratory range (2.6 &mgr;V2; P=0.005). In the TWA range, the change was smaller but highly pronounced relative to the 60- to 120-minute level (65%; P=0.003). The low-frequency and high-frequency heart rate variability power declined before the arrhythmia (P=0.04 and P=0.06, respectively). Conclusions— The magnitude of repolarization instability, manifested by TWA and beat-to-beat oscillations of T-wave amplitudes at other frequencies, increased before the onset of VTA. Tracking of these dynamics can facilitate timely detection of high-risk periods and may be useful for initiation of preventive treatments.

Mouse models of long QT syndrome

Congenital long QT syndrome is a rare inherited condition characterized by prolongation of action potential duration (APD) in cardiac myocytes, prolongation of the QT interval on the surface electrocardiogram (ECG), and an increased risk of syncope and sudden death due to ventricular tachyarrhythmias. Mutations of cardiac ion channel genes that affect repolarization cause the majority of the congenital cases. Despite detailed characterizations of the mutated ion channels at the molecular level, a complete understanding of the mechanisms by which individual mutations may lead to arrhythmias and sudden death requires study of the intact heart and its modulation by the autonomic nervous system. Here, we will review studies of molecularly engineered mice with mutations in the genes (a) known to cause long QT syndrome in humans and (b) specific to cardiac repolarization in the mouse. Our goal is to provide the reader with a comprehensive overview of mouse models with long QT syndrome and to emphasize the advantages and limitations of these models.

Cardiac arrhythmias: from (transgenic) mice to men.

Cardiac Arrhythmias. Transgenic and gene‐targeted mice now are frequently used to study cardiac arrhythmias due to the ease with which the mouse genome can be manipulated. Marked electrophysiologic differences are present between the mouse and human heart, however, and the utility of the mouse as a model for arrhythmias and sudden death remains controversial. Tachyarrhythmias, bradyarrhythmias, and ECG in the mouse need to be interpreted with extreme care and without preconceptions based on our experience with humans. Despite its limitations, the mouse can provide a powerful tool to further our understanding of basic mechanisms that underlie human cardiac electrophysiology.