HCN4编码的I_f在胚胎心室起搏活动中的非必要性
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摘要
目的：
自超极化激活内向电流被发现以来,其特性就被广为研究。但因其作用表现和表达分布的复杂多样性,其在心脏起搏活动中的确切作用一直存在争议。本研究拟从电生理学方面明确超极化激活内向离子通道在早期胚胎心肌细胞上所起的作用。
方法与结果：
1.观察If阻断剂对If，ICaL和ICaT电流的影响
方法：采用胶原酶B消化获得单个小鼠胚胎心肌细胞并培养24-36小时。选择跳动的单个细胞,细胞为E10.5天心肌细胞,1 mMCs+离子作为阻断剂,以全细胞电压钳模式记录心肌细胞的If,IcaL和ICaT电流表达。
结果：lmM Cs+显著抑制If(减少93.1-1.7%,n=13,p<0.05),冲洗后能完全恢复。与此同时,Cs+仅轻度升高L型钙电流(至15.5士4.12%,n--11,p<0.05)。对T型钙电流基本无影响(n=11,p>0.05)。这表明相对L型钙电流和T型钙电流而言,Cs+对If通道存在相对特异阻断性。因此我们在后续实验中采用Cs+作为If通道阻断剂来观察各项目。
2.If在E9.5-E10.5起搏样细胞(P-Iike)起搏活动中所起的作用
方法：采用胶原酶B消化分离以获得单个小鼠胚胎心肌细胞,培养24-36小时后,选择E9.5和E10.5起搏样细胞(P-like)进行研究,应用全细胞电流钳模式以记录E9.5和E10.5起搏样细胞的动作电位,观察比较If在E9.5-E10.5起搏样细胞(P-like)起搏活动中所起的作用。
结果：Cs+在E9.5-E10.5起搏样细胞上引起负性调节效果。此效果在全部的E9.5(n=3)和E10.5(n=3)上均可发现。在E9.5,Cs+使得动作电位频率降低18.7土2.2%(n=3,p>0.05),4期自动除极速率降低21.34-3.1%(n=3)。在E10.5, 1mM Cs+存在和E9.5类似的作用效果,但未发现统计学意义。动作电位频率降低24.6±-2.3%,(p<0.05),4期自动除极速率降低23.9±-3.5%,p>0.05。
3.If在E9.5-E10.5心室样细胞(V-like)起搏活动中所起的作用
方法：采用胶原酶B消化分离以获得单个小鼠胚胎心肌细胞,培养24-36小时后,选择E9.5和E10.5心室样细胞(V-like)进行研究,应用全细胞电流钳模式以记录E9.5和E10.5心室样细胞的动作电位,观察比较If在E9.5-E10.5心室样细胞(V-like)起搏活动中所起的作用。
结果：在81.8%(11个细胞中的9个)E9.5的V-like细胞和27.3%(22个细胞中的6个)的E10.5的V-like细胞上观察到了Cs+的影响。在E9.5的V-like细胞上,Cs+降低动作电位频率70.5±6.9%(n=9,p<0.01),同时降低4期自动除极速率75.6±4.8%(n=6,p<0.05)。在E10.5,Cs+的作用效果要弱于E9.5。Cs+降低动作电位频率65.4±7.2%(p<0.05),同时降低4期自动除极速率75.1±5.9%(p<0.05)。在E17.5也观察到Cs+存在相似的作用效果。
4.If和动作电位的联系
方法：采用胶原酶B消化分离以获得单个小鼠胚胎心肌细胞,培养24-36小时后,选择E8.5和E10.5的成功记录到动作电位的,心房样细胞atrial-like (A-like) cells和心室样细胞及起搏样细胞进行研究。观察这些细胞上是否存在If电流的表达。
结果：E10.5心脏上,HCN4是mRNA水平的主要表达亚型,但Cs+对动作电位的影响仅在一部分细胞上能看到,这可能是由于If的功能表达与mRNA水平并不一致。因此我们观察了存在动作电位的细胞上If电流的表达。在E8.5,If能在91.7%的起搏样细胞,90%的心房样细胞和80%的心室样细胞上检测到。而在E10.5,If存在于70%的起搏样细胞,但只有50%的心房样细胞或心室样细胞上检测到If。以上结果表明,在E10.5的心室肌细胞上,动作电位的产生和If并无直接联系。
结论：
1.在胚胎发育过程中,超极化激活内向电流能稳定持续参与心脏起搏样细胞的起搏活动。
2.在胚胎发育过程中,胚胎心室起搏活动与超极化激活内向电流之间并无必然联系。
目的：
为明确超极化激活内向离子通道作用表现和表达分布的复杂多样性与其亚型表达之间的关系,本研究拟从分子生物学方面明确超极化激活内向离子通道的亚型与其在早期胚胎心肌细胞上所起的作用之间的关系。
方法与结果：
1.HCN亚型表达的发育性变化
方法：采用RT-PCR技术,比较在早期胚胎发育过程中,从E8.5至E10.5,小鼠胚胎心室肌组织HCN2和HCN4亚型在mRNA水平的表达。
结果：早期胚胎发育期中,Cs+对心室细胞起搏活动影响的变化可能归根于基因表达的差异,因此我们检测了HCN2和HCN4亚型在mRNA水平的表达。在胚胎发育过程中,HCN4表达下调而HCN2表达上调。从E8.5至E10.5,HCN4均为心脏的HCN主要表达亚型。虽然HCN2和HCN4改变量并不显著(p>0.05),但己表现出逐步变化的趋势。
2.敲除HCN4对E10.5的心室肌细胞的动作电位和4期自动除极速率的影响方法：采用小干扰核糖核酸技术(siRNA)以观察敲除HCN4基因后对E10.5心室样细胞的影响。
结果：为了观察HCN4-型在E10.5心室肌细胞上的具体功能,我们应用小干扰核糖核酸技术(SiRNA)来敲除HCN4亚型。在敲除组,仅有20%的心室肌细胞检测到If电流,而且If的电流密度从26.0±2.7pA/pF(对照组,n=8)降低至5.6±1.8pA/pF(n=10)(p<0.01,对照组：敲除组)。然而,心室肌细胞的动作电位和4期自动除极速率均未改变(p>0.05)。以上结果表明,尽管HCN4在E10.5的心室肌细胞的mRNA水平显著表达,但HCN4所编码的I_f在胚胎心室肌细胞的起搏活动中所起作用有限。
结论：
1.HCN4确实是早期胚胎心脏上超极化激活阳离子通道的主要表型。
2.超极化激活内向电流的作用除了mRNA表达水平影响以外,可能更重要受到具体亚型功能表达的影响。
3.胚胎心室起搏活动不依赖于HCN4编码的I_f。
Objective
For 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 Results
1. Effects of Cs+ on If, IcaL and ICaT
Methods: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 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 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 APs
Methods: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.
Objective
By 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 Results
1. Developmental Changes in Expression of HCN Subunits
Methods: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 cardiomyocytes
Methods: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.
自超极化激活内向电流被发现以来,其特性就被广为研究。但因其作用表现和表达分布的复杂多样性,其在心脏起搏活动中的确切作用一直存在争议。本研究拟从电生理学方面明确超极化激活内向离子通道在早期胚胎心肌细胞上所起的作用。
方法与结果：
1.观察If阻断剂对If，ICaL和ICaT电流的影响
方法：采用胶原酶B消化获得单个小鼠胚胎心肌细胞并培养24-36小时。选择跳动的单个细胞,细胞为E10.5天心肌细胞,1 mMCs+离子作为阻断剂,以全细胞电压钳模式记录心肌细胞的If,IcaL和ICaT电流表达。
结果：lmM Cs+显著抑制If(减少93.1-1.7%,n=13,p<0.05),冲洗后能完全恢复。与此同时,Cs+仅轻度升高L型钙电流(至15.5士4.12%,n--11,p<0.05)。对T型钙电流基本无影响(n=11,p>0.05)。这表明相对L型钙电流和T型钙电流而言,Cs+对If通道存在相对特异阻断性。因此我们在后续实验中采用Cs+作为If通道阻断剂来观察各项目。
2.If在E9.5-E10.5起搏样细胞(P-Iike)起搏活动中所起的作用
方法：采用胶原酶B消化分离以获得单个小鼠胚胎心肌细胞,培养24-36小时后,选择E9.5和E10.5起搏样细胞(P-like)进行研究,应用全细胞电流钳模式以记录E9.5和E10.5起搏样细胞的动作电位,观察比较If在E9.5-E10.5起搏样细胞(P-like)起搏活动中所起的作用。
结果：Cs+在E9.5-E10.5起搏样细胞上引起负性调节效果。此效果在全部的E9.5(n=3)和E10.5(n=3)上均可发现。在E9.5,Cs+使得动作电位频率降低18.7土2.2%(n=3,p>0.05),4期自动除极速率降低21.34-3.1%(n=3)。在E10.5, 1mM Cs+存在和E9.5类似的作用效果,但未发现统计学意义。动作电位频率降低24.6±-2.3%,(p<0.05),4期自动除极速率降低23.9±-3.5%,p>0.05。
3.If在E9.5-E10.5心室样细胞(V-like)起搏活动中所起的作用
方法：采用胶原酶B消化分离以获得单个小鼠胚胎心肌细胞,培养24-36小时后,选择E9.5和E10.5心室样细胞(V-like)进行研究,应用全细胞电流钳模式以记录E9.5和E10.5心室样细胞的动作电位,观察比较If在E9.5-E10.5心室样细胞(V-like)起搏活动中所起的作用。
结果：在81.8%(11个细胞中的9个)E9.5的V-like细胞和27.3%(22个细胞中的6个)的E10.5的V-like细胞上观察到了Cs+的影响。在E9.5的V-like细胞上,Cs+降低动作电位频率70.5±6.9%(n=9,p<0.01),同时降低4期自动除极速率75.6±4.8%(n=6,p<0.05)。在E10.5,Cs+的作用效果要弱于E9.5。Cs+降低动作电位频率65.4±7.2%(p<0.05),同时降低4期自动除极速率75.1±5.9%(p<0.05)。在E17.5也观察到Cs+存在相似的作用效果。
4.If和动作电位的联系
方法：采用胶原酶B消化分离以获得单个小鼠胚胎心肌细胞,培养24-36小时后,选择E8.5和E10.5的成功记录到动作电位的,心房样细胞atrial-like (A-like) cells和心室样细胞及起搏样细胞进行研究。观察这些细胞上是否存在If电流的表达。
结果：E10.5心脏上,HCN4是mRNA水平的主要表达亚型,但Cs+对动作电位的影响仅在一部分细胞上能看到,这可能是由于If的功能表达与mRNA水平并不一致。因此我们观察了存在动作电位的细胞上If电流的表达。在E8.5,If能在91.7%的起搏样细胞,90%的心房样细胞和80%的心室样细胞上检测到。而在E10.5,If存在于70%的起搏样细胞,但只有50%的心房样细胞或心室样细胞上检测到If。以上结果表明,在E10.5的心室肌细胞上,动作电位的产生和If并无直接联系。
结论：
1.在胚胎发育过程中,超极化激活内向电流能稳定持续参与心脏起搏样细胞的起搏活动。
2.在胚胎发育过程中,胚胎心室起搏活动与超极化激活内向电流之间并无必然联系。
目的：
为明确超极化激活内向离子通道作用表现和表达分布的复杂多样性与其亚型表达之间的关系,本研究拟从分子生物学方面明确超极化激活内向离子通道的亚型与其在早期胚胎心肌细胞上所起的作用之间的关系。
方法与结果：
1.HCN亚型表达的发育性变化
方法：采用RT-PCR技术,比较在早期胚胎发育过程中,从E8.5至E10.5,小鼠胚胎心室肌组织HCN2和HCN4亚型在mRNA水平的表达。
结果：早期胚胎发育期中,Cs+对心室细胞起搏活动影响的变化可能归根于基因表达的差异,因此我们检测了HCN2和HCN4亚型在mRNA水平的表达。在胚胎发育过程中,HCN4表达下调而HCN2表达上调。从E8.5至E10.5,HCN4均为心脏的HCN主要表达亚型。虽然HCN2和HCN4改变量并不显著(p>0.05),但己表现出逐步变化的趋势。
2.敲除HCN4对E10.5的心室肌细胞的动作电位和4期自动除极速率的影响方法：采用小干扰核糖核酸技术(siRNA)以观察敲除HCN4基因后对E10.5心室样细胞的影响。
结果：为了观察HCN4-型在E10.5心室肌细胞上的具体功能,我们应用小干扰核糖核酸技术(SiRNA)来敲除HCN4亚型。在敲除组,仅有20%的心室肌细胞检测到If电流,而且If的电流密度从26.0±2.7pA/pF(对照组,n=8)降低至5.6±1.8pA/pF(n=10)(p<0.01,对照组：敲除组)。然而,心室肌细胞的动作电位和4期自动除极速率均未改变(p>0.05)。以上结果表明,尽管HCN4在E10.5的心室肌细胞的mRNA水平显著表达,但HCN4所编码的I_f在胚胎心室肌细胞的起搏活动中所起作用有限。
结论：
1.HCN4确实是早期胚胎心脏上超极化激活阳离子通道的主要表型。
2.超极化激活内向电流的作用除了mRNA表达水平影响以外,可能更重要受到具体亚型功能表达的影响。
3.胚胎心室起搏活动不依赖于HCN4编码的I_f。
Objective
For 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 Results
1. Effects of Cs+ on If, IcaL and ICaT
Methods: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 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 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 APs
Methods: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.
Objective
By 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 Results
1. Developmental Changes in Expression of HCN Subunits
Methods: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 cardiomyocytes
Methods: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.
引文
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[1]DiFrancesco D. Pacemaker mechanism in cardiac tissue. Annual review of physiology 1994;55:455-472.
[2]Silverman ME, Grove D, Upshaw CB, Jr. Why does the heart beat? The discovery of the electrical system of the heart. Circulation 2006;113:2775-2781.
[3]Huttmann A. [Discovery of the electrical activity and conduction system of the heart]. Wiener klinische Wochenschrift 1992;104:124-129.
[4]Boineau JP, Schuessler RB, Mooney CR, Wylds AC, Miller CB, Hudson RD, et al. Multicentric origin of the atrial depolarization wave:the pacemaker complex. Relation to dynamics of atrial conduction, P-wave changes and heart rate control. Circulation 1978;58:1036-1048.
[5]Schuessler RB, Boineau JP, Wylds AC, Hill DA, Miller CB, Roeske WR. Effect of canine cardiac nerves on heart rate, rhythm, and pacemaker location. The American journal of physiology 1986;250:H630-644.
[6]Noble D. The surprising heart:a review of recent progress in cardiac electrophysiology. The Journal of physiology 1984;353:1-50.
[7]Irisawa H, Brown HF, Giles W. Cardiac pacemaking in the sinoatrial node. Physiological reviews 1993;73:197-227.
[8]Satoh H, Tsuchida K. Comparison of a calcium antagonist, CD-349, with nifedipine, diltiazem, and verapamil in rabbit spontaneously beating sinoatrial node cells. Journal of cardiovascular pharmacology 1993;21:685-692.
[9]Guo J, Ono K, Noma A. A sustained inward current activated at the diastolic potential range in rabbit sino-atrial node cells. The Journal of physiology 1995;483(Pt 1):1-13.
[10]Satoh H. Role of T-type Ca2+ channel inhibitors in the pacemaker depolarization in rabbit sino-atrial nodal cells. General pharmacology 1995;26:581-587.
[11]Mitsuiye T, Shinagawa Y, Noma A. Sustained inward current during pacemaker depolarization in mammalian sinoatrial node cells. Circulation research 2000;87:88-91.
[12]Shinagawa Y, Satoh H, Noma A. The sustained inward current and inward rectifier K+ current in pacemaker cells dissociated from rat sinoatrial node. The Journal of physiology 2000;523 Pt 3:593-605.
[13]Guo J, Mitsuiye T, Noma A. The sustained inward current in sino-atrial node cells of guinea-pig heart. Pflugers Arch 1997;433:390-396.
[14]Satoh H. Sino-atrial nodal cells of mammalian hearts:ionic currents and gene expression of pacemaker ionic channels. Journal of smooth muscle research=Nihon Heikatsukin Gakkai kikanshi 2003;39:175-193.
[15]Robinson RB. Hyperpolarization-activated cation currents:from molecules to function. Annual review of physiology 2003;65:463-480.
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[22]Boyett MR, Honjo H, Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovascular research 2000;47:658-687.
[23]Baruscotti M, Bucchi A, Difrancesco D. Physiology and pharmacology of cardiac pacemaker current. Pharmacology & therapeutics 2005;107:59-69.
[24]DiFrancesco D, Ferroni A, Mazzanti M. Properties of the hyperpolarizing-activated current in cells isolated from rabbit sino-atrial node. The Journal of physiology 1986;377:61-88.
[25]Accili EA, Robinson RB, DiFrancesco D. Properties and modulation of If in newborn versus adult cardiac SA node. The American journal of physiology 1997;272:H1549-1552.
[26]Barbuti A, Baruscotti M, Altomare C, Moroni A, DiFrancesco D. Action of internal pronase on the f-channel kinetics in the rabbit SA node. The Journal of physiology 1999;520 Pt 3:737-744.
[27]Zhou Z, Lipsius SL. Effect of isoprenaline on I(f) current in latent pacemaker cells isolated from cat right atrium:ruptured vs. perforated patch whole-cell recording methods. Pflugers Arch 1993;423:442-447.
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[30]Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER, Siegelbaum SA, et al. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell 1998;93:717-729.
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[37]Ulens C, Tytgat J. Functional heteromerizations of HCN1 and HCN2 pacemaker channels. The Journal of biological chemistry 2001;276:6069-6072.
[38]Schram G, Melnyk P, Nattel S. Differential distributions of cardiac ion channel expression as basis for regional specialization in electrical function. Circulation research 2002;90:939-950.
[39]Moroni A, Barbuti A, Altomare C, Viscomi C, Morgan J, Baruscotti M, et al. Kinetic and ionic properties of the human HCN2 pacemaker channel. Pflugers Arch 2000;439:618-626.
[40]Er F, Ludwig A. Dominant suppression of HCN channels reduces the native pacemaker current and undermines spontaneous beating of neonatal cardiomyocytes. Circulation 2003; 107:485-489..
[41]Ludwig A, Jeglitsch M. The family of the hyperpolarization-activated mammalian cations channels. Nature 1998;393:587-591
[42]Altomare C, Bucchi A, Camatini E. Integrated allosteric model of voltage gating HCN channels. The Journal of general physiology 2001;117:519-532.
[43]Ludwig A, Stieber J, Biel M. The two pacemaker channels from the human heart with different activation kinetics. The EMBO journal 1999;18:2323-2329.
[44]Seifert R, Scholten A, Gauss R, Mincheva A, Lichter P, Kaupp UB. Molecular characterization of a slowly gating human hyperpolarization-activated channel predominantly expressed in thalamus, heart, and testis. Proceedings of the National Academy of Sciences of the United States of America 1999;96:9391-9396.
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[46]Altomare C, Brioschi C. Heteromeric HCN1-HCN4 channels:comparison with native pacemaker channels from rabbit sinoatrial node. The Journal of physiology 2003;549:347-359.
[47]Ishii TM, Takano M, Ohmori H. Determinants in mammalian hyperpolarization-activated cation channels. The Journal of physiology 2001;537:93-100.
[48]Chen J, Mitcheson JS, Tristani-Firouzi M. The S4-S5 linker couples voltage sensing of pacemaker channels. Proceedings of the National Academy of Sciences of the United States of America 2001;98:11277-11282.
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[6]Noble D. The surprising heart:a review of recent progress in cardiac electrophysiology. The Journal of physiology 1984;353:1-50.
[7]Irisawa H, Brown HF, Giles W. Cardiac pacemaking in the sinoatrial node. Physiological reviews 1993;73:197-227.
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[11]Mitsuiye T, Shinagawa Y, Noma A. Sustained inward current during pacemaker depolarization in mammalian sinoatrial node cells. Circulation research 2000;87:88-91.
[12]Shinagawa Y, Satoh H, Noma A. The sustained inward current and inward rectifier K+ current in pacemaker cells dissociated from rat sinoatrial node. The Journal of physiology 2000;523 Pt 3:593-605.
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[22]Boyett MR, Honjo H, Kodama I. The sinoatrial node, a heterogeneous pacemaker structure. Cardiovascular research 2000;47:658-687.
[23]Baruscotti M, Bucchi A, Difrancesco D. Physiology and pharmacology of cardiac pacemaker current. Pharmacology & therapeutics 2005;107:59-69.
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