Novel Mechanisms of Regulation of SK channels: Implications for Cardiac Arrhythmia

摘要 小电导钙激活K+(SK)通道存在于肌膜和线粒体内 心肌细胞膜(IMM、MSK)。它们有一种独特的能力将细胞内 [Ca~(2+)]同时具有质膜复极化和线粒体功能。SK频道，尽管被认为是 在健康中处于休眠状态，与动物模型和人类患者的室性心律失常有关 心衰(HF)。SK通道的异质性上调可加重心律失常的底物。 然而，最近积累的证据表明，在HF或长QT综合征中，SK通道提供 通过减轻复极储备的损失和减少钙离子依赖的心律失常触发因素来保护。 SK通道作为抗心律失常治疗靶点的潜力尚未确定，主要是因为 对控制SK功能的细胞和分子机制缺乏了解。我们的主要目标是 用大鼠肥厚衰竭模型揭示SSK和MSK通道的调节机制 胸主动脉环扎术(TAB)。我们的中心假设是SSK和MSK都是积极的 受丝氨酸-苏氨酸激酶PKA调节，受酪氨酸激酶PYK2负性调节 调节通道对钙/电压依赖性阻滞剂的反应性。我们假设PKA介导的功能 SSKs和MSKs的上调通过减弱肥厚的心律失常电位发挥适应性作用 但是，所提供的保护并不完整。因此，我们认为SSK的进一步增强 和/或MSK活性可以通过抑制Pyk2介导的磷酸化来实现，这可以起到 作为一种减少复极相关心脏病心律失常的新方法 钙离子动态平衡的储备和缺陷，如肥厚和心衰。 本研究的具体目的是：1.通过一种新的研究方法，确定在VCM中SSK功能上调的机制。 TAB大鼠肥厚合并心力衰竭模型。2：确定MSK上调的机制及其在 RyR2对TAB大鼠血管内皮细胞钙释放的调节我们假设MSK上调促进了 线粒体脊扁平，导致三级超复合体(SCS)的形成增加 电子传输链(ETC)元素，从而提高ETC效率和降低MITO-ROS率 产生导致改善细胞内钙动态平衡的。在整个心脏水平上，会导致心律失常 将利用膜的光学映射来研究SK通道的遗传增强或抑制的影响 电位和钙离子；在单细胞水平上，来自TAB心脏的心肌细胞将被结合 新型亚细胞室膜片钳共聚焦显微钙离子和ROS成像 生物传感器、线粒体膜电位和电流、先进的电子断层扫描和生化 接近了。用腺相关病毒载体进行心脏特异性递送将用于修饰表达 TAB心脏中SK通道的水平和靶向。

Abstract Small conductance Ca2+-activated K+ (SK) channels are present in sarcolemma (sSK) and inner mitochondria membrane (IMM, mSK) in ventricular cardiomyocytes (VCMs). They have a unique ability to link intracellular [Ca2+] with both plasmamembrane repolarization and mitochondria function. SK channels, although thought to be dormant in health, are implicated in ventricular arrhythmias in animal models and in human patients with heart failure (HF). Heterogeneous upregulation of SK channels can exacerbate substrate for arrhythmia. However, recently accumulated evidence suggests that in HF or Long QT syndrome, SK channels provide protection by mitigating loss of repolarization reserve and by reducing Ca2+-dependent triggers for arrhythmia. The potential of SK channels as a target for anti-arrhythmic therapy is yet to be determined largely because of the lack of understanding of cellular and molecular mechanisms that govern SK function. Our main objective is to unravel mechanisms of regulation of sSK and mSK channels using rat model of hypertrophy and failure induced by thoracic aortic banding (TAB). Our central hypothesis is that both sSKs and mSKs are positively regulated by the serine-threonine kinase PKA and negatively regulated by the Tyrosine kinase Pyk2 via modulating channel responsiveness to Ca2+/voltage-dependent block. We posit that PKA-mediated functional upregulation of sSKs and mSKs plays an adaptive role by attenuating arrhythmic potential in hypertrophic hearts, but that the protection offered is not complete. We therefore reason that further enhancement of sSK and/or mSK activity can be achieved via inhibition of Pyk2-mediated phosphorylation, and that this could serve as a novel approach to decrease arrhythmias in cardiac diseases associated with reduced repolarization reserve and defective Ca2+ homeostasis such as hypertrophy and HF. The Specific Aims are: 1: To determine the mechanisms of functional upregulation of sSKs in VCMs using a TAB rat model of hypertrophy and HF. 2: To determine the mechanisms of mSK upregulation and their role in regulation of RyR2-mediated Ca2+ release in TAB rat VCMs. We hypothesize that mSK upregulation facilitates mitochondria cristae flattening which leads to increase in formation of tertiary supercomplexes (SCs) from elements of Electron Transport Chain (ETC), thereby enhancing ETC efficiency and reducing rate of mito-ROS production resulting in improvement in intracellular Ca2+ homeostasis. At the whole heart level, arrhythmogenic effects of genetic enhancement or inhibition of SK channels will be studied using optical mapping of membrane potential and Ca2+; at the single cell level, myocytes from TAB hearts will be investigated with a combination of patch clamp, confocal microscopic imaging of Ca2+ and ROS using novel subcellular-compartmental biosensors, mitochondrial membrane potential and currents, advanced electron tomography and biochemical approaches. Cardiac-specific delivery with Adeno-associated viral vectors will be used to modify expression levels and targeting of SK channels in TAB hearts.