Sophie Demolombe

Sodium channel β1 subunit mutations associated with Brugada syndrome and cardiac conduction disease in humans

Brugada syndrome is a genetic disease associated with sudden cardiac death that is characterized by ventricular fibrillation and right precordial ST segment elevation on ECG. Loss-of-function mutations in SCN5A, which encodes the predominant cardiac sodium channel alpha subunit NaV1.5, can cause Brugada syndrome and cardiac conduction disease. However, SCN5A mutations are not detected in the majority of patients with these syndromes, suggesting that other genes can cause or modify presentation of these disorders. Here, we investigated SCN1B, which encodes the function-modifying sodium channel beta1 subunit, in 282 probands with Brugada syndrome and in 44 patients with conduction disease, none of whom had SCN5A mutations. We identified 3 mutations segregating with arrhythmia in 3 kindreds. Two of these mutations were located in a newly described alternately processed transcript, beta1B. Both the canonical and alternately processed transcripts were expressed in the human heart and were expressed to a greater degree in Purkinje fibers than in heart muscle, consistent with the clinical presentation of conduction disease. Sodium current was lower when NaV1.5 was coexpressed with mutant beta1 or beta1B subunits than when it was coexpressed with WT subunits. These findings implicate SCN1B as a disease gene for human arrhythmia susceptibility.

Regional and tissue specific transcript signatures of ion channel genes in the non‐diseased human heart

The various cardiac regions have specific action potential properties appropriate to their electrical specialization, resulting from a specific pattern of ion‐channel functional expression. The present study addressed regionally defined differential ion‐channel expression in the non‐diseased human heart with a genomic approach. High‐throughput real‐time RT‐PCR was used to quantify the expression patterns of 79 ion‐channel subunit transcripts and related genes in atria, ventricular epicardium and endocardium, and Purkinje fibres isolated from 15 non‐diseased human donor hearts. Two‐way non‐directed hierarchical clustering separated atria, Purkinje fibre and ventricular compartments, but did not show specific patterns for epicardium versus endocardium, nor left‐ versus right‐sided chambers. Genes that characterized the atria (versus ventricles) included Cx40, Kv1.5 and Kir3.1 as expected, but also Cav1.3, Cav3.1, Cavα2δ2, Navβ1, TWIK1, TASK1 and HCN4. Only Kir2.1, RyR2, phospholamban and Kv1.4 showed higher expression in the ventricles. The Purkinje fibre expression‐portrait (versus ventricle) included stronger expression of Cx40, Kv4.3, Kir3.1, TWIK1, HCN4, ClC6 and CALM1, along with weaker expression of mRNA encoding Cx43, Kir2.1, KChIP2, the pumps/exchangers Na+,K+‐ATPase, NCX1, SERCA2, and the Ca2+‐handling proteins RYR2 and CASQ2. Transcripts that were more strongly expressed in epicardium (versus endocardium) included Cav1.2, KChIP2, SERCA2, CALM3 and calcineurin‐α. Nav1.5 and Navβ1 were more strongly expressed in the endocardium. For selected genes, RT‐PCR data were confirmed at the protein level. This is the first report of the global portrait of regional ion‐channel subunit‐gene expression in the non‐diseased human heart. Our data point to significant regionally determined ion‐channel expression differences, with potentially important implications for understanding regional electrophysiology, arrhythmia mechanisms, and responses to ion‐channel blocking drugs. Concordance with previous functional studies suggests that regional regulation of cardiac ion‐current expression may be primarily transcriptional.

Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart.

Even though sequencing of the mammalian genome has led to the discovery of a large number of ionic channel genes, identification of the molecular determinants of cellular electrical properties in different regions of the heart has been rarely obtained. We developed a high‐throughput approach capable of simultaneously assessing the expression pattern of ionic channel repertoires from different regions of the mouse heart. By using large‐scale real‐time RT‐PCR, we have profiled 71 channels and related genes in the sinoatrial node (SAN), atrioventricular node (AVN), the atria (A) and ventricles (V). Hearts from 30 adult male C57BL/6 mice were microdissected and RNA was isolated from six pools of five mice each. TaqMan data were analysed using the threshold cycle (Ct) relative quantification method. Cross‐contamination of each region was checked with expression of the atrial and ventricular myosin light chains. Two‐way hierarchical clustering analysis of the 71 genes successfully classified the six pools from the four distinct regions. In comparison with the A, the SAN and AVN were characterized by higher expression of Navβ1, Navβ3, Cav1.3, Cav3.1 and Cavα2δ2, and lower expression of Kv4.2, Cx40, Cx43 and Kir3.1. In addition, the SAN was characterized by higher expression of HCN1 and HCN4, and lower expression of RYR2, Kir6.2, Cavβ2 and Cavγ4. The AVN was characterized by higher expression of Nav1.1, Nav1.7, Kv1.6, Kvβ1, MinK and Cavγ7. Other gene expression profiles discriminate between the ventricular and the atrial myocardium. The present study provides the first genome‐scale regional ionic channel expression profile in the mouse heart.

Conditional Mineralocorticoid Receptor Expression in the Heart Leads to Life-Threatening Arrhythmias

Background—Life-threatening cardiac arrhythmia is a major source of mortality worldwide. Besides rare inherited monogenic diseases such as long-QT or Brugada syndromes, which reflect abnormalities in ion fluxes across cardiac ion channels as a final common pathway, arrhythmias are most frequently acquired and associated with heart disease. The mineralocorticoid hormone aldosterone is an important contributor to morbidity and mortality in heart failure, but its mechanisms of action are incompletely understood. Methods and Results—To specifically assess the role of the mineralocorticoid receptor (MR) in the heart, in the absence of changes in aldosteronemia, we generated a transgenic mouse model with conditional cardiac-specific overexpression of the human MR. Mice exhibit a high rate of death prevented by spironolactone, an MR antagonist used in human therapy. Cardiac MR overexpression led to ion channel remodeling, resulting in prolonged ventricular repolarization at both the cellular and integrated levels and in severe ventricular arrhythmias. Conclusions—Our results indicate that cardiac MR triggers cardiac arrhythmias, suggesting novel opportunities for prevention of arrhythmia-related sudden death.

Human Atrial Ion Channel and Transporter Subunit Gene-Expression Remodeling Associated With Valvular Heart Disease and Atrial Fibrillation

Background—Valvular heart disease (VHD), which often leads to atrial fibrillation (AF), and AF both cause ion-channel remodeling. We evaluated the ion-channel gene expression profile of VHD patients, in permanent AF (AF-VHD) or in sinus rhythm (SR-VHD), in comparison with patients without AF or VHD, respectively. Methods and Results—We used microarrays containing probes for human ion-channel and Ca2+-regulator genes to quantify mRNA expression in atrial tissues from 7 SR-VHD patients and 11 AF-VHD patients relative to 11 control patients in SR without structural heart disease (SR-CAD). From the data set, we selected for detailed analysis 59 transcripts expressed in the human heart. SR-VHD patients differentially expressed 24/59 ion-channel and Ca2+-regulator transcripts. There was significant overlap between VHD groups, with 66% of genes altered in SR-VHD patients being similarly modified in AF-VHD. Statistical differences between the AF- and SR-VHD groups identified the specific molecular portrait of AF, which involved 12 genes that were further confirmed by real-time reverse transcription–polymerase chain reaction. For example, phospholamban, the &bgr;-subunit MinK (KCNE1) and MIRP2 (KCNE3), and the 2-pore potassium channel TWIK-1 were upregulated in AF-VHD compared with SR-VHD, whereas the T-type calcium-channel Cav3.1 and the transient-outward potassium channel Kv4.3 were downregulated. Two-way hierarchical clustering separated SR-VHD from AF-VHD patients. AF-related changes in L-type Ca2+-current and inward-rectifier current were confirmed at protein and functional levels. Finally, for 13 selected genes, SR restoration reversed ion-channel remodeling. Conclusions—VHD extensively remodels cardiac ion-channel and transporter expression, and AF alters ion-channel expression in VHD patients.

Mouse model of SCN5A-linked hereditary Lenegre's disease - Age-related conduction slowing and myocardial fibrosis

Background—We have previously linked hereditary progressive cardiac conduction defect (hereditary Lenègre’s disease) to a loss-of-function mutation in the gene encoding the main cardiac Na+ channel, SCN5A. In the present study, we investigated heterozygous Scn5a-knockout mice (Scn5a+/− mice) as a model for hereditary Lenègre’s disease. Methods and Results—In Scn5a+/− mice, surface ECG recordings showed age-related lengthening of the P-wave and PR- and QRS-interval duration, coinciding with previous observations in patients with Lenègre’s disease. Old but not young Scn5a+/− mice showed extensive fibrosis of their ventricular myocardium, a feature not seen in wild-type animals. In old Scn5a+/− mice, fibrosis was accompanied by heterogeneous expression of connexin 43 and upregulation of hypertrophic markers, including &bgr;-MHC and skeletal &agr;-actin. Global connexin 43 expression as assessed with Western blots was similar to wild-type mice. Decreased connexin 40 expression was seen in the atria. Using pangenomic microarrays and real-time PCR, we identified in Scn5a+/− mice an age-related upregulation of genes encoding Atf3 and Egr1 transcription factors. Echocardiography and hemodynamic investigations demonstrated conserved cardiac function with aging and lack of ventricular hypertrophy. Conclusions—We conclude that Scn5a+/− mice convincingly recapitulate the Lenègre’s disease phenotype, including progressive impairment with aging of atrial and ventricular conduction associated with myocardial rearrangements and fibrosis. Our work provides the first demonstration that a monogenic ion channel defect can progressively lead to myocardial structural anomalies.

Haploinsufficiency in combination with aging causes SCN5A-linked hereditary Lenègre disease

OBJECTIVES The goal of this study was to investigate the genotype-to-phenotype relationship between SCN5A gene mutation and progressive cardiac conduction defect in order to gain insights into the pathophysiologic mechanisms of the disease. BACKGROUND Progressive cardiac conduction defect is a frequent disease commonly attributed to degeneration and fibrosis of the His bundle and its branches. In a French family, we have identified a splicing mutation in the SCN5A gene leading to hereditary progressive cardiac conduction defect. METHODS We have extended the size of the pedigree and phenotyped and genotyped all family members, and also investigated in vitro the functional consequences of the mutation. RESULTS Among 65 potentially affected members, 25 individuals were carriers of the IVS.22+2 T-->C SCN5A mutation. In relation to aging, gene carriers exhibit various types of conduction defects. P-wave, PR, and QRS duration increased progressively with age in gene carriers and in noncarriers. Whatever the age, conduction parameters were longer in gene carriers. The widening in the QRS complex with aging was more pronounced in gene carriers older than 40 years. Functional studies show that the IVS.22+2 T-->C SCN5A mutation lead to exon 22 skipping and to a complete loss of function of the affected allele, but to a normal trafficking of the mutated gene product. CONCLUSIONS Our findings demonstrate that hereditary Lenègre disease is caused by a haploinsufficiency mechanism, which in combination with aging leads to progressive alteration in conduction velocity.

Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease.

The identification of nearly a dozen ion channel genes involved in the genesis of human atrial and ventricular arrhythmias has been critical for the diagnosis and treatment of fatal cardiovascular diseases. In contrast, very little is known about the genetic and molecular mechanisms underlying human sinus node dysfunction (SND). Here, we report a genetic and molecular mechanism for human SND. We mapped two families with highly penetrant and severe SND to the human ANK2 (ankyrin-B/AnkB) locus. Mice heterozygous for AnkB phenocopy human SND displayed severe bradycardia and rate variability. AnkB is essential for normal membrane organization of sinoatrial node cell channels and transporters, and AnkB is required for physiological cardiac pacing. Finally, dysfunction in AnkB-based trafficking pathways causes abnormal sinoatrial node (SAN) electrical activity and SND. Together, our findings associate abnormal channel targeting with human SND and highlight the critical role of local membrane organization for sinoatrial node excitability.

Targeted Deletion of Kv4.2 Eliminates Ito,f and Results in Electrical and Molecular Remodeling, With No Evidence of Ventricular Hypertrophy or Myocardial Dysfunction

Previous studies have demonstrated a role for voltage-gated K+ (Kv) channel &agr; subunits of the Kv4 subfamily in the generation of rapidly inactivating/recovering cardiac transient outward K+ current, Ito,f, channels. Biochemical studies suggest that mouse ventricular Ito,f channels reflect the heteromeric assembly of Kv4.2 and Kv4.3 with the accessory subunits, KChIP2 and Kv&bgr;1, and that Kv4.2 is the primary determinant of regional differences in (mouse ventricular) Ito,f densities. Interestingly, the phenotypic consequences of manipulating Ito,f expression in different mouse models are distinct. In the experiments here, the effects of the targeted deletion of Kv4.2 (Kv4.2−/−) were examined. Unexpectedly, voltage-clamp recordings from Kv4.2−/− ventricular myocytes revealed that Ito,f is eliminated. In addition, the slow transient outward K+ current, Ito,s, and the Kv1.4 protein (which encodes Ito,s) are upregulated in Kv4.2−/− ventricles. Although Kv4.3 mRNA/protein expression is not measurably affected, KChIP2 expression is markedly reduced in Kv4.2−/− ventricles. Similar to Kv4.3, expression of Kv&bgr;1, as well as Kv1.5 and Kv2.1, is similar in wild-type and Kv4.2−/− ventricles. In addition, and in marked contrast to previous findings in mice expressing a truncated Kv4.2 transgene, the elimination Ito,f in Kv4.2−/− mice does not result in ventricular hypertrophy. Taken together, these findings demonstrate not only an essential role for Kv4.2 in the generation of mouse ventricular Ito,f channels but also that the loss of Ito,f per se does not have overt pathophysiological consequences.

Haplotype-Sharing Analysis Implicates Chromosome 7q36 Harboring DPP6 in Familial Idiopathic Ventricular Fibrillation

Idiopathic Ventricular Fibrillation (IVF) is defined as spontaneous VF without any known structural or electrical heart disease. A family history is present in up to 20% of probands with the disorder, suggesting that at least a subset of IVF is hereditary. A genome-wide haplotype-sharing analysis was performed for identification of the responsible gene in three distantly related families in which multiple individuals died suddenly or were successfully resuscitated at young age. We identified a haplotype, on chromosome 7q36, that was conserved in these three families and was also shared by 7 of 42 independent IVF patients. The shared chromosomal segment harbors part of the DPP6 gene, which encodes a putative component of the transient outward current in the heart. We demonstrated a 20-fold increase in DPP6 mRNA levels in the myocardium of carriers as compared to controls. Clinical evaluation of 84 risk-haplotype carriers and 71 noncarriers revealed no ECG or structural parameters indicative of cardiac disease. Penetrance of IVF was high; 50% of risk-haplotype carriers experienced (aborted) sudden cardiac death before the age of 58 years. We propose DPP6 as a gene for IVF and increased DPP6 expression as the likely pathogenetic mechanism.