Flavien Charpentier

Electrophysiologic characteristics of cells spanning the left ventricular wall of human heart: Evidence for presence of M cells

OBJECTIVES The present work was designed to provide an initial characterization of M cells in the normal human heart. BACKGROUND Recent studies have uncovered a unique population of cells in the midmyocardial region of the canine ventricle. These cells, named M cells, were found to possess electrophysiologic features and a pharmacologic responsiveness different from those of other myocardial cells. Although well characterized in the dog, their presence or absence in the human heart is unknown. METHODS Standard microelectrode techniques were used to map slices of ventricular free wall obtained from normal human hearts (n = 4). Preparations were paced at cycle lengths ranging from 1 to 10 s. RESULTS We identified three cell subtypes: endocardial, subepicardial (M cells) and epicardial cells. The principal features differentiating M cells from the other cell subtypes were their longer action potential duration, more accentuated action potential duration rate relations and greater maximal rate of increase in action potential upstroke (Vmax). Our findings suggest that M cells represent approximately 30% of the cellular mass of the left ventricular wall. Concordance between changes in their repolarization and changes in QTU interval provide support for the role of M cells in the generation of the electrocardiographic (ECG) U wave. CONCLUSIONS This study provides evidence for the existence of M cells in the human heart that contribute to heterogeneity of repolarization within the ventricular wall. Our findings provide strong support for the hypothesis that M cells contribute importantly to the manifestation of the U wave on the ECG.

Bradycardia and Slowing of the Atrioventricular Conduction in Mice Lacking CaV3.1/α1G T-Type Calcium Channels

The generation of the mammalian heartbeat is a complex and vital function requiring multiple and coordinated ionic channel activities. The functional role of low-voltage activated (LVA) T-type calcium channels in the pacemaker activity of the sinoatrial node (SAN) is, to date, unresolved. Here we show that disruption of the gene coding for Cav3.1/&agr;1G T-type calcium channels (cacna1g) abolishes T-type calcium current (ICa,T) in isolated cells from the SAN and the atrioventricular node without affecting the L-type Ca2+ current (ICa,L). By using telemetric electrocardiograms on unrestrained mice and intracardiac recordings, we find that cacna1g inactivation causes bradycardia and delays atrioventricular conduction without affecting the excitability of the right atrium. Consistently, no ICa,T was detected in right atrium myocytes in both wild-type and Cav3.1−/− mice. Furthermore, inactivation of cacna1g significantly slowed the intrinsic in vivo heart rate, prolonged the SAN recovery time, and slowed pacemaker activity of individual SAN cells through a reduction of the slope of the diastolic depolarization. Our results demonstrate that Cav3.1/T-type Ca2+ channels contribute to SAN pacemaker activity and atrioventricular conduction.

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.

Impaired Impulse Propagation in Scn5a-Knockout Mice Combined Contribution of Excitability, Connexin Expression, and Tissue Architecture in Relation to Aging

Background—The SCN5A sodium channel is a major determinant for cardiac impulse propagation. We used epicardial mapping of the atria, ventricles, and septae to investigate conduction velocity (CV) in Scn5a heterozygous young and old mice. Methods and Results—Mice were divided into 4 groups: (1) young (3 to 4 months) wild-type littermates (WT); (2) young heterozygous Scn5a-knockout mice (HZ); (3) old (12 to 17 months) WT; and (4) old HZ. In young HZ hearts, CV in the right but not the left ventricle was reduced in agreement with a rightward rotation in the QRS axes; fibrosis was virtually absent in both ventricles, and the pattern of connexin43 (Cx43) expression was similar to that of WT mice. In old WT animals, the right ventricle transversal CV was slightly reduced and was associated with interstitial fibrosis. In old HZ hearts, right and left ventricle CVs were severely reduced both in the transversal and longitudinal direction; multiple areas of severe reactive fibrosis invaded the myocardium, accompanied by markedly altered Cx43 expression. The right and left bundle-branch CVs were comparable to those of WT animals. The atria showed only mild fibrosis, with heterogeneously disturbed Cx40 and Cx43 expression. Conclusions—A 50% reduction in Scn5a expression alone or age-related interstitial fibrosis only slightly affects conduction. In aged HZ mice, reduced Scn5a expression is accompanied by the presence of reactive fibrosis and disarrangement of gap junctions, which results in profound conduction impairment.

Microarray Analysis Reveals Complex Remodeling of Cardiac Ion Channel Expression With Altered Thyroid Status Relation to Cellular and Integrated Electrophysiology

Abstract— Although electrophysiological remodeling occurs in various myocardial diseases, the underlying molecular mechanisms are poorly understood. cDNA microarrays containing probes for a large population of mouse genes encoding ion channel subunits (“IonChips”) were developed and exploited to investigate remodeling of ion channel transcripts associated with altered thyroid status in adult mouse ventricle. Functional consequences of hypo- and hyperthyroidism were evaluated with patch-clamp and ECG recordings. Hypothyroidism decreased heart rate and prolonged QTc duration. Opposite changes were observed in hyperthyroidism. Microarray analysis revealed that hypothyroidism induces significant reductions in KCNA5, KCNB1, KCND2, and KCNK2 transcripts, whereas KCNQ1 and KCNE1 expression is increased. In hyperthyroidism, in contrast, KCNA5 and KCNB1 expression is increased and KCNQ1 and KCNE1 expression is decreased. Real-time RT-PCR validated these results. Consistent with microarray analysis, Western blot experiments confirmed those modifications at the protein level. Patch-clamp recordings revealed significant reductions in Ito,f and IK,slow densities, and increased IKs density in hypothyroid myocytes. In addition to effects on K+ channel transcripts, transcripts for the pacemaker channel HCN2 were decreased and those encoding the &agr;1C Ca2+ channel (CaCNA1C) were increased in hypothyroid animals. The expression of Na+, Cl−, and inwardly rectifying K+ channel subunits, in contrast, were unaffected by thyroid hormone status. Taken together, these data demonstrate that thyroid hormone levels selectively and differentially regulate transcript expression for at least nine ion channel &agr;- and &bgr;-subunits. Our results also document the potential of cDNA microarray analysis for the simultaneous examination of ion channel transcript expression levels in the diseased/remodeled myocardium.

Phosphodiesterase 4B in the cardiac L-type Ca2+ channel complex regulates Ca2+ current and protects against ventricular arrhythmias in mice

β-Adrenergic receptors (β-ARs) enhance cardiac contractility by increasing cAMP levels and activating PKA. PKA increases Ca²⁺-induced Ca²⁺ release via phosphorylation of L-type Ca²⁺ channels (LTCCs) and ryanodine receptor 2. Multiple cyclic nucleotide phosphodiesterases (PDEs) regulate local cAMP concentration in cardiomyocytes, with PDE4 being predominant for the control of β-AR-dependent cAMP signals. Three genes encoding PDE4 are expressed in mouse heart: Pde4a, Pde4b, and Pde4d. Here we show that both PDE4B and PDE4D are tethered to the LTCC in the mouse heart but that β-AR stimulation of the L-type Ca²⁺ current (ICa,L) is increased only in Pde4b-/- mice. A fraction of PDE4B colocalized with the LTCC along T-tubules in the mouse heart. Under β-AR stimulation, Ca²⁺ transients, cell contraction, and spontaneous Ca²⁺ release events were increased in Pde4b-/- and Pde4d-/- myocytes compared with those in WT myocytes. In vivo, after intraperitoneal injection of isoprenaline, catheter-mediated burst pacing triggered ventricular tachycardia in Pde4b-/- mice but not in WT mice. These results identify PDE4B in the CaV1.2 complex as a critical regulator of ICa,L during β-AR stimulation and suggest that distinct PDE4 subtypes are important for normal regulation of Ca²⁺-induced Ca²⁺ release in cardiomyocytes.

Multifocal ectopic Purkinje-related premature contractions: a new SCN5A-related cardiac channelopathy.

OBJECTIVES The aim of this study was to describe a new familial cardiac phenotype and to elucidate the electrophysiological mechanism responsible for the disease. BACKGROUND Mutations in several genes encoding ion channels, especially SCN5A, have emerged as the basis for a variety of inherited cardiac arrhythmias. METHODS Three unrelated families comprising 21 individuals affected by multifocal ectopic Purkinje-related premature contractions (MEPPC) characterized by narrow junctional and rare sinus beats competing with numerous premature ventricular contractions with right and/or left bundle branch block patterns were identified. RESULTS Dilated cardiomyopathy was identified in 6 patients, atrial arrhythmias were detected in 9 patients, and sudden death was reported in 5 individuals. Invasive electrophysiological studies demonstrated that premature ventricular complexes originated from the Purkinje tissue. Hydroquinidine treatment dramatically decreased the number of premature ventricular complexes. It normalized the contractile function in 2 patients. All the affected subjects carried the c.665G>A transition in the SCN5A gene. Patch-clamp studies of resulting p.Arg222Gln (R222Q) Nav1.5 revealed a net gain of function of the sodium channel, leading, in silico, to incomplete repolarization in Purkinje cells responsible for premature ventricular action potentials. In vitro and in silico studies recapitulated the normalization of the ventricular action potentials in the presence of quinidine. CONCLUSIONS A new SCN5A-related cardiac syndrome, MEPPC, was identified. The SCN5A mutation leads to a gain of function of the sodium channel responsible for hyperexcitability of the fascicular-Purkinje system. The MEPPC syndrome is responsive to hydroquinidine.

Delayed rectifier K+ currents and cardiac repolarization

The two components of the cardiac delayed rectifier current have been the subject of numerous studies since firstly described. This current controls the action potential duration and is highly regulated. After identification of the channel subunits underlying IKs, KCNQ1 associated with KCNE1, and IKr, HERG, their involvement in human cardiac channelopathies have provided various models allowing the description of the molecular mechanisms of the KCNQ1 and HERG channels trafficking, activity and regulation. More recently, studies have been focusing on the unveiling of different partners of the pore-forming proteins that contribute to their maturation, trafficking, activity and/or degradation, on one side, and on their respective expression in the heterogeneous cardiac tissue, on the other side. The aim of this review is to report and discuss the major works on IKs and IKr and the most recent ones that help to understand the precise function of these currents in the heart.