锚蛋白B介导M-2受体信号通路调节窦房结功能的机制研究

Sinoatrial nodal (SAN) dysfunction is considered a disease of the elderly with an exponential increase in its frequency with age. Studies have linked ankyrin-B mutation with SAN disease in human and ankyrin-B deficient (Ank-B+/-) mice. We recently found that Ank-B+/- mice showed enhanced negative heart rates (HRs) responses to M2 muscarinic receptors (M2-R) agonist carbachol (CCH) stimulation at the levels of whole body and isolated heart when compared with wild-type (WT) mice, suggesting that ankyrin-B plays an important role in M2-R signaling pathway. Unfortunately, despite the link between ankyrin-B and SAN dysfunction, the molecular mechanisms underlying the interactions of ankyrin-B and M2-R signaling in SAN cells are not completely understood. To further demonstrate the CCH effects on M2-R signaling, we'll check the HRs response to atropine (M2-R antagonist), propranolol (β1-R antagonist) plus CCH as well as adenosine 1 receptor (A1-R) agonist phenylisopropyladenosine (PIA) in vivo and isolated hearts from Ank-B+/- and WT mice. SAN tissues from Ank-B+/- mice will be isolated to measure autonomic never ending distribution and the densities of M1-R, M2-R, and M2-R receptors. CCH binds M2-R on SAN cells, triggering the G protein-gated, inwardly rectifying potassium channel (IKACh) to reduce HRs. IKACh is a heteromultimeric complex formed by Kir3.1 and Kir3.4 subunits. To define molecular determinants of IKACh channel membrane organization, we will assess the Kir3.1 and Kir3.4 cytoplasmic domains for potential targeting motifs. Interestingly, sequence analysis of Kir3.4 revealed striking similarity between a C-terminal cytoplasmic sequence and a motif recently established to target Kir6.2 channels (via ankyrin proteins) to specialized domains of myocytes and pancreas. Further analysis of this motif revealed high sequence conservation between Kir3.4 orthologs. We will evaluate potential interaction of Kir3.4 and ankyrin-B by co-immunoprecipitation and examine a potential direct ankyrin-B/Kir3.4 interaction using purified proteins. Cultured HEK cells and fibroblasts with mutant protein of ankyrin-B will be transfected with normal or mutent kir3.4 plus Kir3.1 to demonstrated the functional expression of IKACh channel. Recent evidence indicates that the membrane clock (mainly hyperpolarization-activated pacemaker current If) and Ca2+ clocks (SR Ca2+ release) jointly regulate SAN automaticity.How ankyrin-B beats these clocks through M2-R signaling was not revealed. As a result, we will measure all of the related currents, including action potentials (APs), diastolic depolarization rate (DDR), If, IKAch, ICa,L, ICa,T , INCX and SR Ca2+ content as well as their response to CCH stimulation in Ank-B+/- SAN cells. We hope to pursue a detailed investingation of how ankyrin-B regulates SAN function through M2-R signaling.

窦房结功能障碍的发生率随年龄增加呈指数上升，其机制仍有待阐明。我们新近发现：毒蕈碱2型（M-2）受体兴奋剂卡巴胆碱可使锚蛋白B基因敲除杂合子（Ank-B+/－）小鼠离体和在体心脏的窦性心率较野生组进一步降低，提示锚蛋白B介导了窦房结的M-2受体信号系统。本研究拟选用Ank-B+/－小鼠检测其心脏电生理特点、副交感神经末梢和M-2受体密度分布情况；分离窦房结细胞测定与M-2受体偶联的乙酰胆碱敏感性钾电流（IKACh）和去敏度，检测卡巴胆碱对涉及起搏机制的If、ICa、 INCX和SR钙负荷的影响；研究锚蛋白B膜结合域与IKACh离子通道亚基Kir3.4之间可能的作用位点，验证其蛋白相互作用；并将正常或突变的Kir3.4分别与正常的Kir3.1表达在含Ank-B+/－的HEK细胞或成纤维细胞，检测IKACh。从而系统阐明锚蛋白B介导M-2受体信号通路调节窦房结功能的分子和电生理机制。

窦房结功能障碍是人类的常见病，而锚蛋白-B缺陷与窦房结功能障碍密切相关，但目前关于锚蛋白-B在窦房结功能障碍中的病理生理作用仍然不清楚。我们的研究发现：终末期缺血性心脏病病人窦房结的锚蛋白-B水平明显降低，而编码乙酰胆碱敏感性钾电流（IKach）的Kir3.1与Kir3.4的蛋白水平明显增加，提示锚蛋白-B可能与IKach电流共同作用而影响窦房结的功能。我们采用锚蛋白-B基因敲除杂合子（Ank-B+/-）小鼠发现：基础状态下Ank-B+/-小鼠的心率显著低于野生型小鼠(P<0.05)， 但使用异丙肾上腺素（ISO）后，Ank-B+/-小鼠的心率净增加数显著大于野生型小鼠(P<0.05)；离体心脏显示两组心率无显著差别，但在50nM的ISO条件下，Ank-B+/-小鼠的心率净增加数显著大于WT小鼠(P<0.05)；细胞水平研究表明Ank-B+/-小鼠窦房结的动作电位（APs）频率显著低于野生型小鼠(P<0.05)，但在ISO条件下频率净增加数显著大于野生型小鼠(P<0.05)，以上结果一致提示Ank-B+/-小鼠对拟交感神经的心率反应增加，其机制是：Ank-B+/-小鼠窦房结细胞的钙负荷（Calcium load）明显降低，在ISO情况下，窦房结细胞钙负荷的净增加量在Ank-B+/-小鼠大于野生型小鼠。此外，腹腔注射兴奋副交感神经的药物卡巴胆碱（CCH)0.1mg/kg后， Ank-B+/-小鼠的心率净降低数较野生型鼠更低(P>0.05)，在不同浓度CCH和基础50nM的ISO条件下，Ank-B+/-小鼠的心率净降低数显著大于WT小鼠(P<0.05)，说明Ank-B+/-小鼠对拟副交感神经刺激的反应也增加。机制研究表明：在10µM CCH的情况下，Ank-B+/-小鼠与野生型小鼠窦房结细胞钙负荷均降低并为同一水平，提示窦房结细胞钙负荷的净降低量在Ank-B+/-小鼠大于野生型小鼠，我们发现Ank-B+/-小鼠的Ikach电流与WT小鼠无显著差别，但其Ikach电流去敏性明显降低，Co-IP实验则证实了Ank-B的膜结构域与Kir3,1（GIRK1）存在蛋白相互作用。Ank-B+/-小鼠的左室有效不应期显著大于WT小鼠(P<0.001)，这可能与Ank-B+/-小鼠表现为多发性心律失常有关。联合干预锚蛋白-B和Kir3.1与Kir3.4可能成为窦房结功能障碍的治疗靶点。