Modulation by bioactive substances of T-type Ca^<2+> channels and their contribution to the regulation of cardiac automaticity

本项目旨在研究t型Ca^<2+>通道在心肌细胞中的功能作用，特别是在窦房结的起搏器细胞中，其与起搏器活性的可能贡献。得到了以下结果：1) t型Ca^<2+>通道与心脏自动性：记录离体豚鼠窦房结细胞的动作电位和膜电流，用膜片钳法研究PGF_<2α>和TXA_2的正向变时作用的离子机制。我们发现PGF_<2α>和TXA_2均增加了离体窦房结细胞的自发放电频率，这种增加是由t型Ca^<2+>通道的激活引起的。结果表明，t型Ca^<2+>通道在PGF_<2α>和txa_2的正性变时作用中具有功能作用。2.2)肺动脉高压时l型和t型Ca^<2+>通道的电重构wistar大鼠注射更多的单碱，导致肺动脉高压伴右心房和心室肥厚。单根碱处理大鼠右心房细胞l型Ca^<2+>通道电流密度显著降低，l型Ca^<2+>通道CaV1.2亚基和副B_2亚基mRNA表达显著降低，B_3亚基mRNA表达显著升高。另一方面，与对照大鼠相比，治疗组大鼠右心房细胞中的t型Ca^<2+>电流更为明显。CaV3.1和CaV3.2 mRNA表达水平及CaV3.1亚基蛋白表达水平均无显著差异。这些结果表明肺动脉高压引起右心房肥厚，与右心房细胞Ca^<2+>通道的电生理分子特性改变有关。3)电压依赖性Ca^<2+>通道的功能分析交感神经末梢肾上腺素的释放依赖于Ca^<2+>通过神经元电压门控n型Ca^<2+>通道进入。Ca^<2+>通道的附属β_3亚基（Cavβ_3）优先与α1B亚基结合形成n型Ca^<2+>通道，因此有望在刺激诱发的去甲肾上腺素释放中发挥功能作用。我们采用Cavβ_3-null、Cavβ_3-过表达（Cavβ_3- tg）和野生型（WT）小鼠，在体内研究了Cavβ_3在交感调节心率中的可能作用，并阐明了Cavβ_3在调节交感神经信号传导中的功能作用。4) TRP通道蛋白的功能分析Ca^<2+>通道在心脏肥厚反应中的重要性已被充分证明，但实际的Ca^<2+>通道仍不清楚。我们证明了TRPC1是心脏肥厚的重要功能调节剂。我们还发现TRPC1在血管平滑肌细胞肥大的发展中起重要作用

This project was carried out to investigate the functional role of the T-type Ca^<2+>channel in cardiac myocytes, particularly in pacemaker cells of sinoatrial node in relation to its possible contribution to the pacemaker activity. The following results were obtained.1) T-type Ca^<2+> channel and cardiac automaticity:The action potential and membrane currents were recorded in isolated guinea-pig sinoatrial node cells and the ionic mechanisms underlying the positive chronotropic action of PGF_<2α> and TXA_2 were investigated by the patch clamp method. We demonstrated that both PGF_<2α> and TXA_2 increased the spontaneous firing frequency of isolated sinoatrial node cells, and that this increase was caused by activation of T-type Ca^<2+> channels. The results indicate the functional role of T-type Ca^<2+> channel in the positive chronotropic action of PGF_<2α> and TXA_2.2) Electrical remodeling of L-and T-type Ca^<2+> Channels during pulmonary hypertensionWistar rats were injected with … More monocrotaline, resulting in pulmonary hypertension with right atrial and ventricular hypertrophy. The L-type Ca^<2+> channel current density was significantly decreased in right atrial cells of monocrotaline-treated rats, accompanied by a significant reduction in mRNA expression of the L-type Ca^<2+> channel CaV1.2 subunit and accessory B_2 subunit, and an increase in the B_3 subunit. On the other hand, T-type Ca^<2+> current was more marked in the right atrial cells of monocrotaline-treated rats than in those of control rats. No significant differences were observed in the mRNA expression levels of CaV3.1 and CaV3.2 or the protein level of the CaV3.1 subunit. These results indicate that pulmonary hypertension causes right atrial hypertrophy, associated with alteration of the electrophysiologic molecular properties of Ca^<2+> channels in right atrial cells.3) Functional analysis of voltage-dependent Ca^<2+> channelsNoradrenaline release from sympathetic nerve terminals is dependent on Ca^<2+> entry through neuronal voltage-gated N-type Ca^<2+> channels. The accessory β_3 subunits of Ca^<2+> channels (Cavβ_3) are preferentially associated with α1B subunit to form N-type Ca^<2+> channels, and are therefore expected to play a functional role in the stimulation-evoked release of noradrenaline. We employed Cavβ_3-null, Cavβ_3-overexpresing (Cavβ_3-Tg), and wild type (WT) mice to investigate the possible roles of Cavβ_3 in the sympathetic regulation of heart rate in vivo, and clarified the functional roles of Cavβ_3 in regulating sympathetic nerve signaling.4) Functional analysis of TRP channel proteinsThe importance of Ca^<2+> entry in the cardiac hypertrophic response is well documented, but the actual Ca^<2+> entry channels remained unknown. We demonstrated TRPC1 as a functionally important regulator of cardiac hypertrophy. We also showed that TRPC1 plays an important role in the development of hypertrophy of vascular smooth muscle cells Less