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

SK 通道调节的新机制：对心律失常的影响

• 批准号: 10424495
• 负责人: Dmitry A Terentyev
• 金额: $ 60.27万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10424495
• 关键词: Animal Model; Arrhythmia; Atrial Fibrillation; Attenuated; Automobile Driving; Biochemical; Biological Assay; Biosensor; Calcium; Calmodulin; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cell membrane; Cells; Chest; Crista ampullaris; Cyclic AMP-Dependent Protein Kinases; Data; Dependovirus; Development; Diabetes Mellitus; Electron Microscope; Electron Transport; Elements; Enhancers; Failure; Feedback; Functional disorder; Genetic; Genetic Enhancement; Health; Heart; Heart Atrium; Heart Diseases; Heart Hypertrophy; Heart failure; Homeostasis; Homologous Gene; Human; Hypertrophy; Image; Inner mitochondrial membrane; Link; Long QT Syndrome; Mediating; Membrane Potentials; Mitochondria; Modeling; Molecular; Muscle Cells; Optics; Pathology; Patients; Phosphorylation; Phosphorylation Site; Play; Potassium; Potassium Channel; Production; Protein Isoforms; Protein Tyrosine Kinase; Protein-Serine-Threonine Kinases; Publishing; Rattus; Reactive Oxygen Species; Regulation; Role; RyR2; Ryanodine Receptor Calcium Release Channel; Sarcolemma; Site-Directed Mutagenesis; Source; Sulfhydryl Compounds; Testing; Therapeutic; Up-Regulation; Ventricular; Ventricular Arrhythmia; Work; adeno-associated viral vector; attenuation; base; electron tomography; experimental study; heart function; insight; microscopic imaging; mitochondrial membrane; novel; novel strategies; overexpression; oxidation; patch clamp; protein kinase A kinase; tomography; voltage

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.

摘要 小电导Ca ~（2+）激活的K ~+（SK）通道存在于肌膜（sSK）和线粒体内 在心室肌细胞（VCMs）中的膜（IMM，mSK）。它们有一种独特的能力， [Ca2+]同时具有质膜复极和线粒体功能。SK频道，虽然被认为 在健康状态下处于休眠状态，与动物模型和人类患者的室性心律失常有关。 心力衰竭（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通道提供了 通过减轻复极储备损失和减少心律失常的Ca 2+依赖性触发来保护。 SK通道作为抗肿瘤治疗靶点的潜力还有待确定，主要是因为 缺乏对控制SK功能的细胞和分子机制的理解。我们的主要目标是 使用大鼠肥大和衰竭模型阐明sSK和mSK通道的调节机制 胸主动脉结扎术（TAB）。我们的中心假设是sSK和mSK都是阳性的， 由丝氨酸-苏氨酸激酶PKA调节，由酪氨酸激酶Pyk 2负调节， 调节通道对Ca 2 +/电压依赖性阻滞的反应性。我们证实PKA介导的功能性 sSKs和mSKs的上调通过减弱肥大细胞的细胞增殖潜能而发挥适应性作用。 心，但提供的保护是不完整的。因此，我们推断，进一步增强sSK 和/或mSK活性可以通过抑制Pyk 2介导的磷酸化来实现，并且这可以用于 作为减少与复极减少相关的心脏疾病中心律失常的新方法 储备和有缺陷的Ca 2+稳态，如肥大和HF。 具体目的是：1.利用免疫组织化学方法，确定VCM中sSK功能上调的机制。 TAB大鼠心肌肥厚和心力衰竭模型。2：确定mSK上调的机制及其在 在TAB大鼠VCM中RyR 2介导的Ca 2+释放的调节。我们假设mSK上调促进了 线粒体嵴变平，导致三级超复合物（SC）的形成增加， 电子传递链（ETC）的元件，从而提高ETC效率并降低线粒体-ROS的速率 产生，导致细胞内Ca 2+稳态的改善。在整个心脏水平， SK通道的遗传增强或抑制的影响将使用膜的光学映射来研究。 电位和Ca 2 +;在单细胞水平，将用以下组合研究来自TAB心脏的肌细胞： 膜片钳，共聚焦显微镜成像的Ca 2+和活性氧使用新的亚细胞间室 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。将使用腺相关病毒载体的心脏特异性递送来修饰表达 TAB心脏中SK通道的水平和靶向。