Changes of HCN gene expression and I(f) currents in Nkx2.5-positive cardiomyocytes derived from murine embryonic stem cells during differentiation.

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, RTPCR, 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.

Subtype switching of T-type Ca 2+ channels from Cav3.2 to Cav3.1 during differentiation of embryonic stem cells to cardiac cell lineage.

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. CONCLUSION: The 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.

State-dependent blocking actions of azimilide dihydrochlo-ride (NE-10064) on human cardiac Na(+) channels.

BACKGROUND: Azimilide reportedly blocks Na(+) channels, although its mechanism remains unclear. METHODS AND RESULTS: The kinetic properties of the azimilide block of the wild-type human Na(+) channels (WT: hH1) and mutant DeltaKPQ Na(+) channels (DeltaKPQ) expressed in COS7 cells were investigated using the whole-cell patch clamp technique and a Markovian state model. Azimilide induced tonic block of WT currents by shifting the h infinity curve in the hyperpolarizing direction and caused phasic block of WT currents with intermediate recovery time constant. The peak and steady-state DeltaKPQ currents were blocked by azimilide, although with only a slight shift in the h infinity curve. The phasic block of peak and steady-state DeltaKPQ currents by azimilide was significantly larger than the blocking of the peak WT current. The affinity of azimilide predicted by a Markovian state model was higher for both the activated state (Kd(A) =1.4 micromol/L), and the inactivated state (Kd(I) =1.4 micromol/L), of WT Na(+) channels than that for the resting state (Kd(R) =102.6 micromol/L). CONCLUSIONS: These experimental and simulation studies suggest that azimilide blocks the human cardiac Na(+) channel in both the activated and inactivated states.

Developmental changes of Ni(2+) sensitivity and automaticity in Nkx2.5-positive cardiac precursor cells from murine embryonic stem cell.

BACKGROUND: It is controversial which subtypes of T type Ca(2+) channels are implicated in automaticity of cardiac cells during the embryonic period. METHOD AND RESULTS: The effect of Ni(2+) on the automaticity of Nkx2.5-positive cardiac precursor cells sorted from embryonic stem cells during their differentiation was examined using patch clamp techniques. Although 40 micromol/L Ni(2+), which is enough to block Ni(2+)sensitive T type-Ca(2+) channels, decreased the spontaneous beating rate in all cells in the early and intermediate stage, Ni(2+) did not show any effects on the automaticity of 50% of the cells in the late stage. CONCLUSION: These results indicate that Ni(2+)-sensitive T-type Ca(2+) channels expressed in the Nkx2.5-positive cardiac precursor cells are developmentally regulated.