ALK4通过PKC/CREB信号通路调控L型钙通道α1C亚基在Ang-II介导的房颤早期电重构及房颤触发的分子机制研究

Abnormal calcium homeostasis in atrial myocytes is one of the most important mechanisms of atrial fibrillation (AF). Increased Ang-II concentration is evident in atrium of patients with AF. Ang-II-induced upregulation of α1C subunit of L-type calcium channel triggers intracellular calcium overload and ultimately affects the triggering and maintenance of AF. In our pilot study, we found that patients with onset AF showed significantly higher Ang-II, ALK4 and CACNA1C expression compared to patients with sinus rhythm. Interestingly, there is a positive correlation of expression of ALK4 and expression of CACNA1C. Further studies show that ALK4 haplodeficiency ameliorates angtiotensin II-induced upregulation of CACNA1C expression, increased calcium current density and increased vulnerability to atrial fibrillation. The above data indicate that ALK4 might be involved in the regulating process of calcium homeostasis of onset AF. In this project, an osmotic mini-pump will be implanted for subcutaneous infusion of Ang-II to induce early atrial electrical remodeling. We will explore the role of ALK4 in Ang-II-induced expression of α1C subunit of L-type calcium channel and calcium homeostasis from the overall, cellular and molecular level. We will focus on PKC/CREB pathway in Ang-II-mediated early electrical remodeling and vulnerability to atrial fibrillation. This study helps clarify the possible mechanism of AF and provides new vision on the optimized therapeutic options based on the mechanistic insights.

心房钙调控异常是房颤发生的重要机制。房颤患者心房肌Ang-II水平升高，L型钙通道α1C亚基基因CACNA1C表达量增加，诱发细胞内钙超载是触发和维持房颤的重要原因。我们前期发现，早期房颤患者心房肌Ang-II、ALK4及CACNA1C表达增加，且CACNA1C与ALK4二者表达量呈正相关。进一步研究发现ALK4表达量减低可抑制Ang-II介导的CACNA1C表达上调、ICa-L密度增加以及房颤的触发。据此我们推测ALK4可能参与房颤钙调控过程。本项目拟在建立Ang-II导致心房电重构模型基础上，采用全细胞膜片钳、荧光素酶报告基因等实验，从整体、细胞及分子水平探讨ALK4对Ang-II介导心房肌CACNA1C表达量及钙调控的影响，明确ALK4影响钙调控的作用及可能机制，重点研究PKC/CREB信号通路在其中发挥的作用。本研究有助于阐明房颤发生的可能机制，为其治疗提供新思路。

目的：房颤是临床最常见的心律失常，可导致卒中和心力衰竭，严重影响患者生活质量。心房钙调控异常是房颤发生的重要机制，本项目拟探讨ALK4对Ang-II介导心房肌CACNA1C表达量及钙调控的影响，明确ALK4影响钙调控的作用及可能机制。.方法：入选因二尖瓣关闭不全行单纯二尖瓣置换术的患者，根据患者的房颤病程及心律分为早期房颤组（房颤病程不超过3个月）和窦性心律组，手术时留取外周静脉血和左心耳组织。通过野生型和ALK4+/-小鼠皮下埋植Ang-II微量渗透压泵构建小鼠早期心房电重构模型，7天后小鼠经心腔内程序性电刺激诱发房颤并留取心房组织。上述患者和小鼠心房组织标本分别检测ALK4、CACNA1C的蛋白表达。分离野生型和ALK4+/-乳鼠心肌细胞，经Ang-II（1μM）刺激后检测各组细胞CACNA1C的转录及蛋白表达，检测心肌细胞L型钙电流，胞浆内Ca2+浓度，钙瞬变及钙调控相关蛋白表达。利用荧光素酶报告基因检测，确定并验证Ang-II调控CACNA1C转录的关键启动子区域。.结果：我们发现早期房颤患者心房组织中ALK4及CACNA1C基因的蛋白表达量明显增加，且二者呈明显正相关。动物和细胞实验均证实ALK4增强Ang-II介导的CACNA1C基因的转录和蛋白表达。利用荧光素酶报告基因检测，通过逐级缩短CACNA1C基因编码区上游启动子区序列，我们将Ang-II促进CACNA1C转录活性增加的核心启动子区定位在-1853bp~-1845bp的CRE序列，并确定CREB是调控转录的关键转录因子。ALK4基因敲除可通过抑制CREB的磷酸化水平，减弱Ang-II对CACNA1C转录活性的调控作用，进而减少CACNA1C的蛋白表达，进而调控L型钙电流触发钙释放过程，最终抑制Ang-II介导的钙调控紊乱，减轻Ang-II介导的AF早期电重构及房颤触发。.结论：Ang-II可促进ALK4/CREB信号通路的激活，转录因子CREB磷酸化水平增加可增强CACNA1C基因的转录水平，继而心房肌细胞L型钙电流密度增加、促进钙触发钙释放过程，钙瞬变增加并最终促进房颤的发生。ALK4基因敲除可以抑制Ang-II介导的上述过程，最终改善Ang-II介导的房颤早期电重构及房颤触发。