Lucía Núñez

Functional Effects of KCNE3 Mutation and Its Role in the Development of Brugada Syndrome

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 (Ito) is thought to play a prominent role in the expression of the syndrome, mutations in Ito-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 Ito intensity compared with WT KCNE3+KCND3. Using tissues isolated from the left atrial appendages of human hearts, we also demonstrate that Kv4.3 and KCNE3 can be coimmunoprecipitated. Conclusions— These results provide definitive evidence for a functional role of KCNE3 in the modulation of Ito in the human heart and suggest that mutations in KCNE3 can underlie the development of the Brugada syndrome.

Insights into genotype–phenotype correlation in hypertrophic cardiomyopathy. Findings from 18 Spanish families with a single mutation in MYBPC3

Background Mutations in the cardiac myosin-binding protein C (MYBPC3) gene are frequently found as a cause of hypertrophic cardiomyopathy (HCM). However, only a few studies have analysed genotype–phenotype correlations in small series of patients. The present study sought to determine the clinical characteristics, penetrance and prognosis of HCM with an identical mutation in MYBPC3. Methods 154 non-related patients with HCM (aged 55±16 years, 100 (64.9%) males) were studied. 18 (11.7%) were found to have an identical mutation in the MYBPC3 gene (IVS23+1G→A). Pedigree analysis, including both clinical evaluation and genotyping, was performed. Results 152 individuals (mean age 37±18 years, 53.3% males) from 18 families were evaluated. 65 carriers of the IVS23+1G→A mutation were identified, 61.5% of whom met HCM diagnostic criteria. Penetrance of the disease increased with age, with 50% affected at 46 years of age. Males tended to develop the disease earlier than females. 7 (15.6%) had systolic dysfunction. Compared with the rest of the HCM cohort, probands with the mutation had more hypertrophy and were younger at diagnosis. There was a trend towards a reduced survival free from sudden death (SD) (HR 1.71; 95% CI 0.98 to 2.98, p=0.059). There were 17 SD cases in 12 families with the mutation. Conclusions The MYBPC3 IVS23+1G→A mutation is associated with middle-age onset disease and poor outcome, with a significant proportion of patients developing systolic impairment and a high SD risk profile.

Nitric oxide inhibits Kv4.3 and human cardiac transient outward potassium current (Ito1).

AIMS Chronic atrial fibrillation (CAF) is characterized by a shortening of the plateau phase of the action potentials (AP) and a decrease in the bioavailability of nitric oxide (NO). In this study, we analysed the effects of NO on Kv4.3 (I(Kv4.3)) and on human transient outward K(+) (I(to1)) currents as well as the signalling pathways responsible for them. We also analysed the expression of NO synthase 3 (NOS3) in patients with CAF. METHODS AND RESULTS I(Kv4.3) and I(to1) currents were recorded in Chinese hamster ovary cells and in human atrial and mouse ventricular dissociated myocytes using the whole-cell patch clamp. The expression of NOS3 was analysed by western blotting. AP were recorded using conventional microelectrode techniques in mouse atrial preparations. NO and NO donors inhibited I(Kv4.3) and human I(to1) in a concentration- and voltage-dependent manner (IC(50) for NO: 375.0 +/- 48 nM) as a consequence of the activation of adenylate cyclase and the subsequent activation of the cAMP-dependent protein kinase and the serine-threonine phosphatase 2A. The density of the I(to1) recorded in ventricular myocytes from wild-type (WT) and NOS3-deficient mice (NOS3(-/-)) was not significantly different. Furthermore, the duration of atrial AP repolarization in WT and NOS3(-/-) mice was not different. The increase in NO levels to 200 nM prolonged the plateau phase of the mouse atrial AP and lengthened the AP duration measured at 20 and 50% of repolarization of the human atrial CAF-remodelled AP as determined using a mathematical model. However, the expression of NOS3 was not modified in left atrial appendages from CAF patients. CONCLUSION Our results suggested that the increase in the atrial NO bioavailability could partially restore the duration of the plateau phase of CAF-remodelled AP by inhibiting the I(to1) as a result of the activation of non-canonical enzymatic pathways.

Spironolactone and its main metabolite canrenoic acid block hKv1.5, Kv4.3 and Kv7.1 + minK channels

Both spironolactone (SP) and its main metabolite, canrenoic acid (CA), prolong cardiac action potential duration and decrease the Kv11.1 (HERG) current. We examined the effects of SP and CA on cardiac hKv1.5, Kv4.3 and Kv7.1+minK channels that generate the human IKur, Ito1 and IKs, which contribute to the control of human cardiac action potential duration. hKv1.5 currents were recorded in stably transfected mouse fibroblasts and Kv4.3 and Kv7.1+minK in transiently transfected Chinese hamster ovary cells using the whole‐cell patch clamp. SP (1 μM) and CA (1 nM) inhibited hKv1.5 currents by 23.2±3.2 and 18.9±2.7%, respectively, shifted the midpoint of the activation curve to more negative potentials and delayed the time course of tail deactivation. SP (1 μM) and CA (1 nM) inhibited the total charge crossing the membrane through Kv4.3 channels at +50 mV by 27.1±6.4 and 27.4±5.7%, respectively, and accelerated the time course of current decay. CA, but not SP, shifted the inactivation curve to more hyperpolarised potentials (Vh−37.0±1.8 vs −40.8±1.6 mV, n=10, P<0.05). SP (10 μM) and CA (1 nM) also inhibited Kv7.1+minK currents by 38.6±2.3 and 22.1±1.4%, respectively, without modifying the voltage dependence of channel activation. SP, but not CA, slowed the time course of tail current decay. CA (1 nM) inhibited the IKur (29.2±5.5%) and the Ito1 (16.1±3.9%) recorded in mouse ventricular myocytes and the IK (21.8±6.9%) recorded in guinea‐pig ventricular myocytes. A mathematical model of human atrial action potentials demonstrated that K+ blocking effects of CA resulted in a lengthening of action potential duration, both in normal and atrial fibrillation simulated conditions. The results demonstrated that both SP and CA directly block hKv1.5, Kv4.3 and Kv7.1+minK channels, CA being more potent for these effects. Since peak free plasma concentrations of CA ranged between 3 and 16 nM, these results indicated that blockade of these human cardiac K+ channels can be observed after administration of therapeutic doses of SP. Blockade of these cardiac K+ currents, together with the antagonism of the aldosterone proarrhythmic effects produced by SP, might be highly desirable for the treatment of supraventricular arrhythmias.

Screening mutations in myosin binding protein C3 gene in a cohort of patients with Hypertrophic Cardiomyopathy

BackgroundMyBPC3 mutations are amongst the most frequent causes of hypertrophic cardiomyopathy, however, its prevalence varies between populations. They have been associated with mild and late onset disease expression. Our objectives were to establish the prevalence of MyBPC3 mutations and determine their associated clinical characteristics in our patients.MethodsScreening by Single Strand Conformation Polymorphisms (SSCP) and sequencing of the fragments with abnormal motility of the MyBPC3 gene in 130 unrelated consecutive HCM index cases. Genotype-Phenotype correlation studies were done in positive families.Results16 mutations were found in 20 index cases (15%): 5 novel [D75N, V471E, Q327fs, IVS6+5G>A (homozygous), and IVS11-9G>A] and 11 previously described [A216T, R495W, R502Q (2 families), E542Q (3 families), T957S, R1022P (2 families), E1179K, K504del, K600fs, P955fs and IVS29+5G>A]. Maximum wall thickness and age at time of diagnosis were similar to patients with MYH7 mutations [25(7) vs. 27(8), p = 0.16], [46(16) vs. 44(19), p = 0.9].ConclusionsMutations in MyBPC3 are present in 15% of our hypertrophic cardiomyopathy families. Severe hypertrophy and early expression are compatible with the presence of MyBPC3 mutations. The genetic diagnosis not only allows avoiding clinical follow up of non carriers but it opens new possibilities that includes: to take preventive clinical decisions in mutation carriers than have not developed the disease yet, the establishment of genotype-phenotype relationship, and to establish a genetic diagnosis routine in patients with familial HCM.

Effects of MiRP1 and DPP6 β‐subunits on the blockade induced by flecainide of KV4.3/KChIP2 channels

The human cardiac transient outward potassium current (Ito) is believed to be composed of the pore‐forming KV4.3 α‐subunit, coassembled with modulatory β‐subunits as KChIP2, MiRP1 and DPP6 proteins. β‐Subunits can alter the pharmacological response of Ito; therefore, we analysed the effects of flecainide on KV4.3/KChIP2 channels coassembled with MiRP1 and/or DPP6 β‐subunits.

Genetically engineered mice as a model for studying cardiac arrhythmias.

Sudden cardiac death due to ventricular tachyarrhythmias remains an unresolved problem, probably because the mechanisms responsible for the progression of cardiac disease to electrophysiological failure are poorly understood. Genetically engineered mice, the principal mammalian model for studying cardiac electrophysiology, have contributed to the understanding of the genetic, molecular and systemic mechanisms involved in the initiation and/or maintenance of cardiac arrhythmias leading to cardiac death, e.g. cardiac excitability, conduction velocity and refractoriness. Several murine models harbouring human gene mutations leading to electrical and structural cardiac disorders have been developed, including channelopathies (long QT syndrome), familial conduction disorders, cardiomyopathies and other inherited cardiac disorders. This article reviews the results of the main genetically modified mice addressing the genesis of cardiac arrhythmias and sudden cardiac death.

Functional characterization of a novel frameshift mutation in the C-terminus of the Nav1.5 channel underlying a Brugada syndrome with variable expression in a Spanish family.

Introduction We functionally analyzed a frameshift mutation in the SCN5A gene encoding cardiac Na+ channels (Nav1.5) found in a proband with repeated episodes of ventricular fibrillation who presented bradycardia and paroxysmal atrial fibrillation. Seven relatives also carry the mutation and showed a Brugada syndrome with an incomplete and variable expression. The mutation (p.D1816VfsX7) resulted in a severe truncation (201 residues) of the Nav1.5 C-terminus. Methods and Results Wild-type (WT) and mutated Nav1.5 channels together with hNavβ1 were expressed in CHO cells and currents were recorded at room temperature using the whole-cell patch-clamp. Expression of p.D1816VfsX7 alone resulted in a marked reduction (≈90%) in peak Na+ current density compared with WT channels. Peak current density generated by p.D1816VfsX7+WT was ≈50% of that generated by WT channels. p.D1816VfsX7 positively shifted activation and inactivation curves, leading to a significant reduction of the window current. The mutation accelerated current activation and reactivation kinetics and increased the fraction of channels developing slow inactivation with prolonged depolarizations. However, late INa was not modified by the mutation. p.D1816VfsX7 produced a marked reduction of channel trafficking toward the membrane that was not restored by decreasing incubation temperature during cell culture or by incubation with 300 μM mexiletine and 5 mM 4-phenylbutirate. Conclusion Despite a severe truncation of the C-terminus, the resulting mutated channels generate currents, albeit with reduced amplitude and altered biophysical properties, confirming the key role of the C-terminal domain in the expression and function of the cardiac Na+ channel.

Efectos del óxido nítrico sobre la función cardíaca

El oxido nitrico (NO) liberado por practicamente todas las celulas del corazon ejerce multiples efectos sobre la funcion cardiaca. Modula las respuestas inotropicas y cronotropicas, el flujo de entrada de Ca ++ y el ciclo del Ca ++ en el reticulo sarcoplasmico, la transmision autonomica, la frecuencia cardiaca, la respiracion mitocondrial, el consumo miocardico de O 2 y la eficiencia mecanica. El NO regula la contractilidad cardiaca en respuesta a la distension e inhibe la relacion fuerza-frecuencia y las respuesta a la estimulacion β-adrenergica. Tambien mejora la distensibilidad ventricular y aumenta el trabajo latido en pacientes con miocardiopatia dilatada, y desempena un importante papel en la fase tardia del precondicionamiento isquemico. Por ultimo, el NO puede modular la actividad de los canales cardiacos, la arritmogenesis, la apoptosis y la funcion cardiaca en el miocardio insuficiente. Para realizar todas estas funciones, las NO sintasas (NOS) se localizan en microdominios de los cardiomiocitos en intima vecindad con las vias de senalizacion que modulan. Sin embargo, es necesario conocer mejor los mecanismos implicados en la regulacion y la localizacion celular de las NOS, asi como las vias no enzimaticas de sintesis del NO, su localizacion y su inactivacion en diversas situaciones fisipatologicas antes de que podamos trasladar las multiples acciones del NO en una alternative terapeutica.

Farmacología de los ácidos grasos omega-3

El consumo de acidos grasos omega-3, como el acido eicosapentanoico (EPA) y el acido docosahexanoico (DHA), derivados de alimentos marinos y de plantas ha demostrado, en estudios epidemiologicos y clinicos, que reduce la incidencia de mortalidad coronaria y la muerte por arritmias. Recientemente, un suplemento que contiene una concentracion del 90% de acidos omega-3 (EPA y DHA) en forma de etil esteres (Omacor ® ) ha sido autorizado como tratamiento adjunto a la dieta para reducir la hipertrigliceridemia en pacientes adultos y, tambien, como tratamiento adjunto a la dieta y a otros tratamientos en la prevencion secundaria del infarto de miocardio. En este articulo, en primer lugar, revisamos la estructura quimica, las acciones farmacologicas y los mecanismos por los cuales los acidos grasos n-3 y, en particular, el Omacor ® , pueden reducir el riesgo de muerte cardiovascular. A continuacion, se analizan las propiedades farmacocineticas, la seguridad y las recomendaciones de diversos organismos para administrar suplementos de EPA+DHA u Omacor ® en los pacientes con enfermedades cardiovasculares.