| ObjectiveFor decades the If channel reveals unique features that are believed to be prerequisite for pacemaker activity. However, If is present in both automatic and nonautomatic regions of the heart, there have been considerable debate over the exact role of the current. By using the whole cell patch clamp technique, we studied the Electrophysiological significance of If for action potential (AP) generation in early developmental fetal cardiomyocytes (E9.5-E10.5).Methods and Results1. Effects of Cs+ on If, IcaL and ICaTMethods:The fetal murine hearts were dissociated to get single cardiomyocytes and cultured for 24-36 h. If, IcaL and IcaT currents were recorded using whole cell voltage patch clamp technique with specific inhibitors including 1 mM Cs+.Results:The main inward currents contributing to diastolic depolarization are If, IcaL and IcaT. To confirm the special effects of Cs+ on If, the side effects of Cs+on IcaL and IcaT were excluded. 1mM Cs+ significantly inhibited If (by 93.1±1.7%, n=13, p<0.05) whereas only slightly increased IcaL (by 15.5±4.12%, n=11, p<0.05). No change in IcaT (n=11, p>0.05) was observed. This indicates that Cs+ has specific effects on inwardly If current while not on IcaL or ICaT.Thus we applied Cs+ as the specific blocker of If in the following investigations.2. Contribution of If to Pacemaker Activity of Pacemaker-like (P-like) Cells from E9.5-E10.5 Hearts Methods:The fetal murine hearts were dissociated to get single cardiomyocytes and cultured for 24-36 h. Whole cell current clamp technique was used to investigate action potential on pacemaker-like cells at E9.5 and E10.5.Results:Cs+ exerted negative chronotropic effect on P-like cells from E9.5-E10.5 hearts. The effect was detected in all P-like cells at E9.5 (n=3) and E10.5 (n=3). At E9.5, Cs+ had negative effect on AP frequency (by 18.7±2.2%, n=3, p>0.05) and Vdd (by 21.3±3.1%, n=3). At E10.5, 1mM Cs+ exerted similar negative chronotropic effect on P-like APs as at E9.5. Although not statistical different, the AP frequency decreased (by 24.6±2.3%, p<0.05) parallely with the Vdd decrease (by 23.9±3.5%, p>0.05).3. Contributions of If to Pacemaker Activity of Ventricular-like (V-like) Cells from E9.5-E10.5 HeartsMethods:The fetal murine hearts were dissociated to get single cardiomyocytes and cultured for 24-36 h. Whole cell current clamp technique was used to investigate action potential on ventricular-like cells at E9.5 and E10.5.Results:The effect of Cs+ was detected in 81.8%(9 out of 11 cells) V-like cells at E9.5 and 27.3%(6 out of 22 cells) V-like cells at E10.5. At E9.5, Cs+ had negative effect on AP frequency in V-like cardiomyocytes (by 70.5±6.9%, n=9, p<0.01). The Vdd decreased by a similar extent (by 75.6±4.8%, n=6, p<0.05). At E10.5, Cs+ 1mM had weaker negative chronotropic effect on V-like APs than that at E9.5. Cs+ 1mM decreased the AP frequency and Vdd by 65.4±7.2%(p<0.05) and 75.1±5.9% (p<0.05), respectively. Similar effects of Cs+ were found in E17.5 ventricular cardiomyocytes (data not shown).4. Correlation of If to APsMethods:The fetal murine hearts were dissociated to get single cardiomyocytes and cultured for 24-36 h. If currents were recorded using whole cell voltage patch clamp technique at cells which exhibited spontaneous APs at E8.5 and E10.5.Results:The whole cell If current was recorded in cells which exhibited spontaneous APs. At E8.5, If was detectable in 91.7% of P-like cells,90% of atrial-like (A-like) cells, and 80% of V-like cells. At E10.5, If was detectable in 70% of P-like cells, while only in 50% of A-like or V-like cells. This observation revealed that there was no exact correlation of If to AP generation in E10.5 ventricular cardiomyocytes.Conclusion:During development, the If current had consistent contribution to pacemaker activity of P-like cardiomyocytes. But for working cardiomyocytes, If current was not necessary for the spontaneous electrical activity. ObjectiveBy using Reverse transcription polymerase chain reaction (RT-PCR) and small interference RNA (SiRNA) technique, we studied the molecularbiological characterizations of If in early developmental fetal cardiomyocytes (E9.5-E10.5).Methods and Results1. Developmental Changes in Expression of HCN SubunitsMethods:The fetal murine hearts were dissociated to get single cardiomyocytes and cultured for 24-36 h. RT-PCR technique was used to investigate the molecular basis of the If electrophysiological properties at mRNA level.Results:The developmental changes in Cs+ effects on pacemaker activity of V-like cells in early developmental stage might aroused from the difference in gene expression, therefore the expression of HCN2 and HCN4 subunits at mRNA level were further investigated. During embryonic development, HCN4 transcripts were downregulated while HCN2 was upregulated. From E8.5 to E10.5, HCN4 was the predominant subunit in the hearts. Both HCN2 and HCN4 did not change significantly (p>0.05), although there was a trend that both of them stepwise changed.2. Knock-down of HCN4 Subunit Influencing neither Vdd nor AP Frequency in E10.5 Ventricular cardiomyocytesMethods:The fetal murine hearts were dissociated to get single cardiomyocytes and cultured for 24-36 h. SiRNA technique was used to investigate the effection of HCN4 Subunit in E10.5 Ventricular cardiomyocytes.Results:To examine the functional role of HCN4 transcripts in E10.5 ventricular cardiomyocytes, SiRNA technique was applied to knock down the HCN4 subunit. In the knock-down group, If was detected in 20% of ventricular cardiomyocytes and the If current density decreased from 26.0±2.7 pA/pF (control, n=8) to 5.6±1.8 pA/pF (n=10) (p<0.01 control vs. knock-down). Interestingly, the Vdd and AP frequency didn’t change (p>0.05). This result indicated that HCN4-encoded If was not involved in the pacemaker activity of fetal ventricular cardiomyocytes despite of its marked expression.Conclusion:At early development stage, the HCN4 transcripts were predominantly expressed. But for working cardiomyocytes, HCN4 encoded If current was not necessary for the spontaneous electrical activity. |
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