Marjan van Kempen

Distribution of connexin37, connexin40 and connexin43 in the aorta and coronary artery of several mammals

Abstract Intercellular communication between cells of the vessel wall is established by a combination of diffusion and convection of humoral and endothelial factors in the extracellular fluid or by direct intercellular contacts present in the form of gap junctions composed of proteins called connexins. At least connexin (Cx)37, Cx40 and Cx43 are expressed in the vessel wall, but disparate findings with regard to the cell specific localisation of connexins in the vasculature indicate that the distribution of connexins may be species and vessel specific. Moreover, differences in expression exist between cells in culture and tissue sections. We performed an inventory immunohistochemical study on the localisation of Cx37, Cx40 and Cx43 on tissue sections of the bovine, micropig and rat aorta and coronary system, which represent morphologically and functionally different types of vessels in the arterial system. We could observe Cx40 labelling most commonly, although with various intensities, between endothelial and smooth muscle cells of the species studied, with the exception of rat aortic smooth muscle cells. The distribution of Cx43 is more differentiated and mostly confined to smooth muscle cells, although it can be detected scarcely between endothelial cells. Cx37, when detectable, is predominantly expressed between endothelial cells in a heterogeneous pattern. We conclude that Cx40 is the constitutive vascular gap junction protein in situ and guarantees cell coupling between cells in the vessel wall. The differentiated distribution of both Cx37 and Cx43 suggests they are involved in more dynamic processes.

Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution

OBJECTIVE The sinoatrial (SA) node consists of a relatively small number of poorly coupled cells. It is not well understood how these pacemaker cells drive the surrounding atrium and at the same time are protected from its hyperpolarizing influence. To explore this issue on a small tissue scale we studied the activation pattern of the mouse SA node region and correlated this pattern with the distribution of different gap junction proteins, connexin (Cx)37, Cx40, Cx43 and Cx45. METHODS AND RESULTS The mouse SA node was electrophysiologically mapped using a conventional microelectrode technique. The primary pacemaker area was located in the corner between the lateral and medial limb of the crista terminalis. Unifocal pacemaking occurred in a group of pacemaking fibers consisting of 450 cells. In the nodal area transitions of nodal and atrial waveform were observed over small distances ( approximately 100 microm). Correlation between the activation pattern and connexin distribution revealed extensive labeling by anti-Cx45 in the primary and secondary pacemaker area. Within these nodal areas no gradient in Cx45 labeling was found. A sharp transition was found between Cx40- and Cx43-expressing myocytes of the crista terminalis and the Cx45-expressing myocytes of the node. In addition, strands of myocytes labeled for Cx43 and Cx40 protrude into the nodal area. Cx37 labeling was only present between endothelial cells. Furthermore, a band of connective tissue largely separates the nodal from the atrial tissue. CONCLUSIONS Our results demonstrate strands of Cx43 and Cx40 positive atrial cells protruding into the Cx45 positive nodal area and a band of connective tissue largely separating the nodal and atrial tissue. This organization of the mouse SA node provides a structural substrate that both shields the nodal area from the hyperpolarizing influence of the atrium and allows fast action potential conduction from the nodal area into the surrounding atrium.

Characterization of Gap Junction Channels in Adult Rabbit Atrial and Ventricular Myocardium

For effective cardiac output, it is essential that electrical excitation spread rapidly throughout the atria and ventricles. This is effected by electrical coupling through gap junction channels at contact sites between myocytes. These channels form a low-resistance pathway between adjacent myocytes and consist of connexin proteins. The connexin family is a large multigene family, and the channels formed by different members of this family have distinct electrical and regulatory properties. We have studied gap junction channels between adult rabbit atrial and ventricular myocytes using immunocytochemical and electrophysiological methods. Gap junctions of ventricular myocytes were immunoreactive to antibodies directed against connexin43 (Cx43) and Cx45, but not to antibodies against Cx37 or Cx40. Gap junctions between atrial myocytes showed immunostaining with anti-Cx40, -Cx43, and -Cx45 antibodies, but not with anti-Cx37 antibody. Endocardial and endothelial tissue were labeled with both Cx37 and Cx40 antibodies. The conductance of rabbit myocardial gap junctions was measured using the double whole-cell voltage-clamp method. The average macroscopic junctional conductance, corrected for series resistance, of atrial and ventricular cell pairs did not differ significantly (169+/-146 and 175+/-147 nS, respectively), and both were at most only slightly sensitive to the applied transjunctional potential difference. The difference in connexin expression between atrial and ventricular myocytes was reflected in the distribution of single gap junction channel conductances. A single population of unitary channel conductances with an average of 100 pS was observed between ventricular myocyte pairs. In addition to this population, a population with an average conductance of 185 pS was present between atrial myocyte pairs. The observed difference in connexin expression between atrial and ventricular myocardium may enable differential regulation of conduction in these tissues.

P19 embryonal carcinoma cells: a suitable model system for cardiac electrophysiological differentiation at the molecular and functional level

Objective: Murine P19 embryonal carcinoma (EC) cells can differentiate into spontaneously beating cardiomyocytes in vitro and have revealed important insight into the early molecular processes of cardiomyocyte differentiation. We assessed the suitability of the P19 cell model for studying cardiac ion channel regulation at the molecular and functional level. Methods: P19 cells were induced to differentiate towards cardiomyocytes. mRNAs for cardiac markers and ion channels were determined by RT-PCR at six timepoints during the differentiation process. Action potentials and individual ion currents were measured by whole cell patch clamp. Results: Ion channel mRNA expression of several channels is temporally regulated during differentiation, while others show little or no regulation. L-type calcium and transient outward channels are expressed from very early on, while sodium and delayed and inward rectifier channels are upregulated at somewhat later stages during differentiation, which mirrors the in vivo murine cardiomyocyte differentiation during embryogenesis. Spontaneous cardiomyocyte action potentials exhibit a low upstroke velocity, which often can be enhanced by hyperpolarizing the cells, hence activating thusfar dormant ion channels to contribute to the action potential upstroke. Action potential duration decreases considerably during the differentiation of spontaneously beating cells. In late stages, non-beating myocytes can be found which only generate action potentials upon electrical stimulation. Their shape is comparable to neonatal/juvenile ventricular mouse myocytes in culture. Finally, we show that P19-derived cardiomyocytes display a very complete set of functional ion channels. Conclusion: P19 cells represent a powerful model to study the regulation of myocardial electrophysiological differentiation at the molecular and functional level.

Genetic and phenotypic heterogeneity suggest therapeutic implications in SCN2A-related disorders

Mutations in SCN2A, a gene encoding the voltage-gated sodium channel Nav1.2, have been associated with a spectrum of epilepsies and neurodevelopmental disorders. Here, we report the phenotypes of 71 patients and review 130 previously reported patients. We found that (i) encephalopathies with infantile/childhood onset epilepsies (≥3 months of age) occur almost as often as those with an early infantile onset (<3 months), and are thus more frequent than previously reported; (ii) distinct phenotypes can be seen within the late onset group, including myoclonic-atonic epilepsy (two patients), Lennox-Gastaut not emerging from West syndrome (two patients), and focal epilepsies with an electrical status epilepticus during slow sleep-like EEG pattern (six patients); and (iii) West syndrome constitutes a common phenotype with a major recurring mutation (p.Arg853Gln: two new and four previously reported children). Other known phenotypes include Ohtahara syndrome, epilepsy of infancy with migrating focal seizures, and intellectual disability or autism without epilepsy. To assess the response to antiepileptic therapy, we retrospectively reviewed the treatment regimen and the course of the epilepsy in 66 patients for which well-documented medical information was available. We find that the use of sodium channel blockers was often associated with clinically relevant seizure reduction or seizure freedom in children with early infantile epilepsies (<3 months), whereas other antiepileptic drugs were less effective. In contrast, sodium channel blockers were rarely effective in epilepsies with later onset (≥3 months) and sometimes induced seizure worsening. Regarding the genetic findings, truncating mutations were exclusively seen in patients with late onset epilepsies and lack of response to sodium channel blockers. Functional characterization of four selected missense mutations using whole cell patch-clamping in tsA201 cells-together with data from the literature-suggest that mutations associated with early infantile epilepsy result in increased sodium channel activity with gain-of-function, characterized by slowing of fast inactivation, acceleration of its recovery or increased persistent sodium current. Further, a good response to sodium channel blockers clinically was found to be associated with a relatively small gain-of-function. In contrast, mutations in patients with late-onset forms and an insufficient response to sodium channel blockers were associated with loss-of-function effects, including a depolarizing shift of voltage-dependent activation or a hyperpolarizing shift of channel availability (steady-state inactivation). Our clinical and experimental data suggest a correlation between age at disease onset, response to sodium channel blockers and the functional properties of mutations in children with SCN2A-related epilepsy.

Remarkable Phenytoin Sensitivity in 4 Children with SCN8A-related Epilepsy: A Molecular Neuropharmacological Approach

Mutations in SCN8A are associated with epilepsy and intellectual disability. SCN8A encodes for sodium channel Nav1.6, which is located in the brain. Gain-of-function missense mutations in SCN8A are thought to lead to increased firing of excitatory neurons containing Nav1.6, and therefore to lead to increased seizure susceptibility. We hypothesized that sodium channel blockers could have a beneficial effect in patients with SCN8A-related epilepsy by blocking the overactive Nav1.6 and thereby counteracting the effect of the mutation. Herein, we describe 4 patients with a missense SCN8A mutation and epilepsy who all show a remarkably good response on high doses of phenytoin and loss of seizure control when phenytoin medication was reduced, while side effects were relatively mild. In 2 patients, repeated withdrawal of phenytoin led to the reoccurrence of seizures. Based on the findings in these patients and the underlying molecular mechanism we consider treatment with (high-dose) phenytoin as a possible treatment option in patients with difficult-to-control seizures due to an SCN8A mutation.

Mutations in KCNT1 cause a spectrum of focal epilepsies

Autosomal dominant mutations in the sodium‐gated potassium channel subunit gene KCNT1 have been associated with two distinct seizure syndromes, nocturnal frontal lobe epilepsy (NFLE) and malignant migrating focal seizures of infancy (MMFSI). To further explore the phenotypic spectrum associated with KCNT1, we examined individuals affected with focal epilepsy or an epileptic encephalopathy for mutations in the gene. We identified KCNT1 mutations in 12 previously unreported patients with focal epilepsy, multifocal epilepsy, cardiac arrhythmia, and in a family with sudden unexpected death in epilepsy (SUDEP), in addition to patients with NFLE and MMFSI. In contrast to the 100% penetrance so far reported for KCNT1 mutations, we observed incomplete penetrance. It is notable that we report that the one KCNT1 mutation, p.Arg398Gln, can lead to either of the two distinct phenotypes, ADNFLE or MMFSI, even within the same family. This indicates that genotype–phenotype relationships for KCNT1 mutations are not straightforward. We demonstrate that KCNT1 mutations are highly pleiotropic and are associated with phenotypes other than ADNFLE and MMFSI. KCNT1 mutations are now associated with Ohtahara syndrome, MMFSI, and nocturnal focal epilepsy. They may also be associated with multifocal epilepsy and cardiac disturbances.

Targeted sequencing of 351 candidate genes for epileptic encephalopathy in a large cohort of patients

Many genes are candidates for involvement in epileptic encephalopathy (EE) because one or a few possibly pathogenic variants have been found in patients, but insufficient genetic or functional evidence exists for a definite annotation.

Nav1.1 dysfunction in genetic epilepsy with febrile seizures-plus or Dravet syndrome

Relatively few SCN1A mutations associated with genetic epilepsy with febrile seizures‐plus (GEFS+) and Dravet syndrome (DS) have been functionally characterized. In contrast to GEFS+, many mutations detected in DS patients are predicted to have complete loss of function. However, functional consequences are not immediately apparent for DS missense mutations. Therefore, we performed a biophysical analysis of three SCN1A missense mutations (R865G, R946C and R946H) we detected in six patients with DS. Furthermore, we compared the functionality of the R865G DS mutation with that of a R859H mutation detected in a GEFS+ patient; the two mutations reside in the same voltage sensor domain of Nav1.1. The four mutations were co‐expressed with β1 and β2 subunits in tsA201 cells, and characterized using the whole‐cell patch clamp technique. The two DS mutations, R946C and R946H, were nonfunctional. However, the novel voltage sensor mutants R859H (GEFS+) and R865G (DS) produced sodium current densities similar to those in wild‐type channels. Both mutants had negative shifts in the voltage dependence of activation, slower recovery from inactivation, and increased persistent current. Only the GEFS+ mutant exhibited a loss of function in voltage‐dependent channel availability. Our results suggest that the R859H mutation causes GEFS+ by a mixture of biophysical defects in Nav1.1 gating. Interestingly, while loss of Nav1.1 function is common in DS, the R865G mutation may cause DS by overall gain‐of‐function defects.

Functional analysis of novel KCNQ2 mutations found in patients with Benign Familial Neonatal Convulsions

Benign Familial Neonatal Convulsions (BFNC) are a rare epilepsy disorder with an autosomal-dominant inheritance. It is linked to mutations in the potassium channel genes KCNQ2 and KCNQ3. These encode for Kv7.2 and Kv7.3 potassium ion channels, which produce an M-current that regulates the potential firing action in neurons through modulation of the membrane potential. We report on the biophysical and biochemical properties of V589X, T359K and P410fs12X mutant-KCNQ2 ion channels that were detected in three BFNC families. Mutant KCNQ2 cDNAs were co-expressed with WT-KCNQ2 and KCNQ3 cDNAs in HEK293 cells to mimic heterozygous expression of the KCNQ2 mutations in BFNC patients. The resulting potassium currents were measured using patch-clamp techniques and showed an approximately 75% reduction in current and a depolarized shift in the voltage dependence of activation. Furthermore, the time-constant of activation of M-currents in cells expressing T359K and P410fs12X was slower compared to cells expressing only wild-type proteins. Immunofluorescent labeling of HEK293 cells stably expressing GFP-tagged KCNQ2-WT or mutant alpha-subunits indicated cell surface expression of WT, V589X and T359K mutants, suggesting a loss-of-function, while P410fs12X was predominantly retained in the ER and sub-cellular compartments outside the ER suggesting an effectively haplo-insufficient effect.