Background The developmental changes of Ni 2+ -sensitivity to automaticity of Nkx2.5-positive cells derived from mouse embryonic stem cell have been identified, suggesting developmental regulation of expressing Ni 2+ -sensitive T-type Ca 2+ channel, although the mechanism of the change has not been fully studied. Methods and Results Transcripts of Cav3.2, Cav3.1 and Cav1.2 genes of beating Nkx2.5-positive cells, which encode the Ni 2+ -sensitive T-type Ca 2+ channel, Ni 2+ -insensitive T-type Ca 2+ channel, and L-type Ca 2+ channel, respectively, were investigated by real-time reverse-transcriptase-polymerase chain reaction, and the current density of each channel was measured by patch-clamp techniques at the early and late stages of differentiation. The expression of the Cav3.2 transcript predominated in the early stage whereas those of Cav3.1 and Cav1.2 transcripts were upregulated in the late stage, which was consistent with the change in each current density, suggesting the expression of channel proteins is largely determined at the transcriptional level. ConclusionThe results indicate that the mechanism of change of Ni 2+ -sensitivity is partly, if not completely, the subtype switch of T-type Ca 2+ channel from Cav3.2 to Cav3.1 at the transcriptional level, and that the expression of the L-type Ca 2+ channel might have an attenuating effect on Ni 2+ -sensitivity to automaticity in the late stage of differentiation. (Circ J 2005; 69: 1284 -1289

No abstract

Changes in the expression of hyperpolarization-activated cyclic nucleotide (HCN)-gated channels and I f currents during the differentiation of embryonic stem cells into cardiac cells remain unknown. We examined changes of HCN genes in expression and function during the differentiation of Nkx2.5-positive cardiac precursor cells derived from mouse ES cells using cell sorting, RT-PCR, immunofluorescence and whole cell patch-clamp techniques. Cs + -induced inhibition of automaticity and transcription of HCN genes increased during differentiation. Expressions of Nkx2.5, a marker of cardiac progenitor cell, and Flk1, a marker of hemangioblast, were mutually exclusive. Messenger RNA and proteins encoded by HCN1 and 4 genes were predominantly observed in Nkx2.5-positive cells on day 15, although Flk1-positive cells did not express genes of the HCN family on that day. Cs + -induced prolongation of the cycle of spontaneous action potentials and I f currents were predominantly observed on day 15. These results suggested that a fraction of Nkx2.5-positive cardiac precursor cells was committed to pacemaking cells expressing I f channels predominantly encoded by HCN 1 and 4 genes.Hyperpolarization-activated cyclic nucleotide-gated channels (I f channels) encoded by a family of four HCN genes (HCN1-4) are widely expressed in heart cells (3) and neurons (21). I f channels are best known for their prime role in the generation of automaticity in cardiac pacemaker cells in the adult sinus node and Purkinje system (3,6,17). Rabbit cardiac sinoatrial nodal cells express HCN1 mRNA together with strongly expressed HCN4 mRNA (7, 16) and rabbit Purkinje fibers contain almost equal amounts of HCN1 and HCN4 transcripts together with a small amount of HCN2 mRNA (23).

zimilide dihydrochloride (NE-10064), a chlorphenylfranyl compound, prolongs cardiac refractoriness by blocking the fast (IKr) and slow (IKs) components of the delayed-rectifier potassium currents. [1][2][3] These in vitro electrophysiologic properties of azimilide could account for its antiarrhythmic and antifibrillatory actions in a number of animal models, which could be classified as class III antiarrhythmic actions. [4][5][6][7][8][9][10] It has been reported that azimilide interacts with other cardiac ion channels, blocking the L-type Ca 2+ channel, the inward rectifier K + channel, and the Na + channel, 11-13 but so far there have not been any systematic studies of the effects of azimilide on the cardiac Na + channels and little is known about the statedependent azimilide block of the Na + channels, although a few reports indicated that azimilide blocked cardiac peak Circulation Journal Vol. 68, July 2004 and late Na + currents. 12-15 Therefore, we assessed the effects of azimilide on the Na + current using the wild-type -subunit of the human cardiac sodium channel (hH1: WT) as well as long QT syndrome type III mutant (∆KPQ mutant) expressed in COS7 cells. Methods Plasmid and ExpressionThe vector pRC/CMV (Invitrogen, San Diego, CA, USA) was used as the expression vector for human WT or ∆KPQ mutant cDNAs. COS7 cells were maintained in Dulbecco's modified Eagle medium (DMEM; Gibco BRL, Gaithersburg, MD, USA)/10% fetal bovine serum, 1% penicillin and 1% streptomycin at 37°C in a 5% CO2 incubator. Cells grown on glass coverslips were co-transfected with both pRC/WT or pRC/∆KPQ mutant and pEGFPC1 (Clontech, Palo Alto, CA, USA) by using Lipofectamine (Gibco BRL) according to the manufacturer's instructions. Forty-eight h after transfection, cells were visualized by EGFP fluorescence and subjected to the whole-cell patch clamp experiments. 16 Electrophysiological RecordingsWhole-cell patch clamp experiments were performed at 22°C. The external solution had the following composition ( mol/L): NaCl 140, CsCl 5.0, MgCl2 1.0, CaCl2 1.8, HEPES 10 and glucose 10, pH 7.4 with NaOH. Patch clamp electrodes were filled with a solution of the following composition ( mol/L): CsF 90, CsCl 10, EGTA 10, NaF 10, MgCl2 2 and HEPES 10, pH 7.4 with CsOH.Currents were recorded with a patch clamp amplifier (Axopatch). The membrane current was filtered at 10 kHz