Céline Marionneau

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.

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.

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.

Distinct Cellular and Molecular Mechanisms Underlie Functional Remodeling of Repolarizing K+ Currents With Left Ventricular Hypertrophy

Left ventricular hypertrophy (LVH) is associated with electric remodeling and increased arrhythmia risk, although the underlying mechanisms are poorly understood. In the experiments here, functional voltage-gated (Kv) and inwardly rectifying (Kir) K+ channel remodeling was examined in a mouse model of pressure overload-induced LVH, produced by transverse aortic constriction (TAC). Action potential durations (APDs) at 90% repolarization in TAC LV myocytes and QTc intervals in TAC mice were prolonged. Mean whole-cell membrane capacitance (Cm) was higher, and Ito,f, IK,slow, Iss, and IK1 densities were lower in TAC, than in sham, LV myocytes. Although the primary determinant of the reduced current densities is the increase in Cm, IK,slow amplitudes were decreased and Iss amplitudes were increased in TAC LV cells. Further experiments revealed regional differences in the effects of LVH. Cellular hypertrophy and increased Iss amplitudes were more pronounced in TAC endocardial LV cells, whereas IK,slow amplitudes were selectively reduced in TAC epicardial LV cells. Consistent with the similarities in Ito,f and IK1 amplitudes, Kv4.2, Kv4.3, and KChIP2 (Ito,f), as well as Kir2.1 and Kir2.2 (IK1), transcript and protein expression levels were similar in TAC and sham LV. Unexpectedly, expression of IK,slow channel subunits Kv1.5 and Kv2.1 was increased in TAC LV. Biochemical experiments also demonstrated that, although total protein was unaltered, cell surface expression of TASK1 was increased in TAC LV. Functional changes in repolarizing K+ currents with LVH, therefore, result from distinct cellular (cardiomyocyte enlargement) and molecular (alterations in the numbers of functional channels) mechanisms.

Structure–Activity Relationships of Human Urotensin II and Related Analogues on Rat Aortic Ring Contraction

The sequence of human urotensin II (UII) has been recently established as H-Glu-Thr-Pro-Asp-Cys-Phe-Trp-Lys-Tyr-Cys-Val-OH, and it has been reported that UII is the most potent mammalian vasoconstrictor peptide identified so far. A series of UII analogues was synthesized, and the contractile activity of each compound was studied in vitro using de-endothelialised rat aortic rings. Replacement of each amino acid by an l-alanine or by a d-isomer showed that the N- and C-terminal residues flanking the cyclic region of the amidated peptide were relatively tolerant to substitution. Conversely, replacement of any residue of the cyclic region significantly reduced the contractile activity of the molecule. The octapeptide UII(4–11) was 4 times more potent than UII, indicating that the C-terminal region of the molecule possesses full biological activity. Alanine or d-isomer substitutions in UII(4–11) or in UII(4–11)-NH2, respectively, showed a good correlation with the results obtained for UII-NH2. Disulfide bridge disruption or replacement of the cysteine residues by their d-enantiomers markedly reduced the vasoconstrictor effect of UII and its analogues. In contrast, acetylation of the N-terminal residue of UII and UII-NH2 enhanced the potency of the peptide. Finally, monoiodination of the Tyr6 residue in UII(4–11) increased by 5 fold the potency of the peptide in the aortic ring bioassay. This structure–activity relationship study should provide useful information for the rational design of selective and potent UII receptor agonists and antagonists.

Calmodulin Kinase II Inhibition Shortens Action Potential Duration by Upregulation of K+ Currents

The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by elevated intracellular Ca2+ (Ca2+i), and mice with chronic myocardial CaMKII inhibition (Inh) resulting from transgenic expression of a CaMKII inhibitory peptide (AC3-I) unexpectedly showed action potential duration (APD) shortening. Inh mice exhibit increased L-type Ca2+ current (ICa), because of upregulation of protein kinase A (PKA) activity, and decreased CaMKII-dependent phosphorylation of phospholamban (PLN). We hypothesized that CaMKII is a molecular signal linking Ca2+i to repolarization. Whole cell voltage-clamp recordings revealed that the fast transient outward current (Ito,f) and the inward rectifier current (IK1) were selectively upregulated in Inh, compared with wild-type (WT) and transgenic control, mice. Breeding Inh mice with mice lacking PLN returned Ito,f and IK1 to control levels and equalized the APD and QT intervals in Inh mice to control and WT levels. Dialysis of AC3-I into WT cells did not result in increased Ito,f or IK1, suggesting that enhanced cardiac repolarization in Inh mice is an adaptive response to chronic CaMKII inhibition rather than an acute effect of reduced CaMKII activity. Increasing PKA activity, by cell dialysis with cAMP, or inhibition of PKA did not affect IK1 in WT cells. Dialysis of WT cells with cAMP also reduced Ito,f, suggesting that PKA upregulation does not increase repolarizing K+ currents in Inh mice. These findings provide novel in vivo and cellular evidence that CaMKII links Ca2+i to cardiac repolarization and suggest that PLN may be a critical CaMKII target for feedback regulation of APD in ventricular myocytes.

The sodium channel accessory subunit Navβ1 regulates neuronal excitability through modulation of repolarizing voltage-gated K+ channels

The channel pore-forming α subunit Kv4.2 is a major constituent of A-type (IA) potassium currents and a key regulator of neuronal membrane excitability. Multiple mechanisms regulate the properties, subcellular targeting, and cell-surface expression of Kv4.2-encoded channels. In the present study, shotgun proteomic analyses of immunoprecipitated mouse brain Kv4.2 channel complexes unexpectedly identified the voltage-gated Na+ channel accessory subunit Navβ1. Voltage-clamp and current-clamp recordings revealed that knockdown of Navβ1 decreases IA densities in isolated cortical neurons and that action potential waveforms are prolonged and repetitive firing is increased in Scn1b-null cortical pyramidal neurons lacking Navβ1. Biochemical and voltage-clamp experiments further demonstrated that Navβ1 interacts with and increases the stability of the heterologously expressed Kv4.2 protein, resulting in greater total and cell-surface Kv4.2 protein expression and in larger Kv4.2-encoded current densities. Together, the results presented here identify Navβ1 as a component of native neuronal Kv4.2-encoded IA channel complexes and a novel regulator of IA channel densities and neuronal excitability.

Homeostatic regulation of electrical excitability in physiological cardiac hypertrophy

Pathological biomechanical stresses cause cardiac hypertrophy, which is associated with QT prolongation and arrhythmias. Previous studies have demonstrated that repolarizing K+ current densities are decreased in pressure overload‐induced left ventricular hypertrophy, resulting in action potential and QT prolongation. Cardiac hypertrophy also occurs with exercise training, but this physiological hypertrophy is not associated with electrical abnormalities or increased arrhythmia risk, suggesting that repolarizing K+ currents are upregulated, in parallel with the increase in myocyte size, to maintain normal cardiac function. To explore this hypothesis directly, electrophysiological recordings were obtained from ventricular myocytes isolated from two mouse models of physiological hypertrophy, one produced by swim‐training of wild‐type mice and the other by cardiac‐specific expression of constitutively active phosphoinositide‐3‐kinase‐p110α (caPI3Kα). Whole‐cell voltage‐clamp recordings revealed that repolarizing K+ current amplitudes were higher in ventricular myocytes isolated from swim‐trained and caPI3Kα, compared with wild‐type, animals. The increases in K+ current amplitudes paralleled the observed cellular hypertrophy, resulting in normalized or increased K+ current densities. Electrocardiographic parameters, including QT intervals, as well as ventricular action potential waveforms in swim‐trained animals/myocytes were indistinguishable from controls, demonstrating preserved electrical function. Additional experiments revealed that inward Ca2+ current amplitudes/densities were also increased in caPI3Kα, compared with WT, left ventricular myocytes. The expression of transcripts encoding K+, Ca2+ and other ion channel subunits was increased in swim‐trained and caPI3Kα ventricles, in parallel with the increase in myocyte size and with the global increases in total cellular RNA expression. In contrast to pathological hypertrophy, therefore, the functional expression of repolarizing K+ (and depolarizing Ca2+) channels is increased with physiological hypertrophy, reflecting upregulation of the underlying ion channel subunit transcripts and resulting in increased current amplitudes and the normalization of current densities and action potential waveforms. Taken together, these results suggest that activation of PI3Kα signalling preserves normal myocardial electrical functioning and could be protective against the increased risk of arrhythmias and sudden death that are prevalent in pathological cardiac hypertrophy.

Variable Na(v)1.5 protein expression from the wild-type allele correlates with the penetrance of cardiac conduction disease in the Scn5a(+/-) mouse model.

Background Loss-of-function mutations in SCN5A, the gene encoding Nav1.5 Na+ channel, are associated with inherited cardiac conduction defects and Brugada syndrome, which both exhibit variable phenotypic penetrance of conduction defects. We investigated the mechanisms of this heterogeneity in a mouse model with heterozygous targeted disruption of Scn5a (Scn5a +/− mice) and compared our results to those obtained in patients with loss-of-function mutations in SCN5A. Methodology/Principal Findings Based on ECG, 10-week-old Scn5a +/− mice were divided into 2 subgroups, one displaying severe ventricular conduction defects (QRS interval>18 ms) and one a mild phenotype (QRS≤18 ms; QRS in wild-type littermates: 10–18 ms). Phenotypic difference persisted with aging. At 10 weeks, the Na+ channel blocker ajmaline prolonged QRS interval similarly in both groups of Scn5a +/− mice. In contrast, in old mice (>53 weeks), ajmaline effect was larger in the severely affected subgroup. These data matched the clinical observations on patients with SCN5A loss-of-function mutations with either severe or mild conduction defects. Ventricular tachycardia developed in 5/10 old severely affected Scn5a +/− mice but not in mildly affected ones. Correspondingly, symptomatic SCN5A–mutated Brugada patients had more severe conduction defects than asymptomatic patients. Old severely affected Scn5a +/− mice but not mildly affected ones showed extensive cardiac fibrosis. Mildly affected Scn5a +/− mice had similar Nav1.5 mRNA but higher Nav1.5 protein expression, and moderately larger INa current than severely affected Scn5a +/− mice. As a consequence, action potential upstroke velocity was more decreased in severely affected Scn5a +/− mice than in mildly affected ones. Conclusions Scn5a +/− mice show similar phenotypic heterogeneity as SCN5A-mutated patients. In Scn5a +/− mice, phenotype severity correlates with wild-type Nav1.5 protein expression.