Electrophysiology of Neonatal and Adult Heart

新生儿和成人心脏电生理学

• 批准号: 8207206
• 负责人: MARTIN MORAD
• 金额: $ 33.17万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8207206
• 关键词: ATP phosphohydrolase; Abbreviations; Acidosis; Adult; Apoptosis; Caffeine; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cardiac; Cardiac Myocytes; Cardiomyopathies; Caveolins; Cells; Characteristics; Confocal Microscopy; Cytoskeletal Proteins; Dependence; Development; Dihydropyridine Receptors; Dyes; Dystrophin; Electrophysiology (science); Embryo; Energy Supply; Family Felidae; Fluorescence; Generations; Heart; Heart Atrium; Human; Hypoxia; ITPR1 gene; Image; Individual; Inositol; Integrins; Isoproterenol; Kinetics; Lactic Acidosis; Leigh Disease; Length; Liquid substance; Location; Manufactured football; Mechanics; Mediating; Membrane Potentials; Metabolic; Mitochondria; Molecular; Monitor; Mus; Muscle Cells; Myocardium; Myopathy; N-Methyl-D-Aspartate Receptors; Nature; Neonatal; Nitric Oxide; Nuclear; Pathology; Peripheral; Physiologic pulse; Play; Process; Production; Proteins; Rattus; Reactive Oxygen Species; Reducing Agents; Reperfusion Injury; Research; Resolution; Role; Ryanodine Receptor Calcium Release Channel; Ryanodine Receptors; Sarcolemma; Sarcoplasmic Reticulum; Signal Pathway; Signal Transduction; Solutions; Source; Spatial Distribution; Staining method; Stains; Stimulus; Stress; Stretching; Structural Protein; Techniques; Testing; Time; Ventricular; Voltage-Dependent Anion Channel; Work; Workload; base; extracellular; fluorescence imaging; improved; infancy; inhibitor/antagonist; meetings; mitochondrial membrane; mitochondrial permeability transition pore; novel; postnatal; pressure; public health relevance; receptor; response; rhod 2-AM; rhod-2; sensor; tempol; tripolyphosphate; voltage clamp

DESCRIPTION (provided by applicant): The long term objective of this project is to characterize the multiplicity of Ca2+ -signaling in normal and abnormal cardiac function. This research is focused on a novel mitochondrial Ca2+-signaling pathway that is triggered by exposing cardiac cells to biologically relevant jets of 'pressurized fluid' (PF). Considering that the strength of the normal heartbeat is mainly determined by Ca2+-induced Ca2+ release (CICR) from the SR, the PF-triggered response may add fine control and plasticity to interaction of CICR with mitochondrial energy production which may become critical in various pressure-flow related myopathies. Our recent results show that PF pulses activate relatively slow (~1.5 s) Ca2+ transients that originate from a small intracellular Ca2+ pool that appear to be independent of transmembrane entry or release of SR Ca2+, nitric oxide (NO) or IP3-signaling pathways, but depend on the integrity of the mitochondrial function as metabolic uncoupler FCCP or mitochondrial Ca2+ uniporter (mCU) blocker, Ru360 suppress them. Reducing agent DTT and reactive oxygen species (ROS) scavenger Tempol, as well as the mitochondrial NCX (mNCX) blocker CGP-37157, also specifically inhibit these Ca2+ transients. In Rhod-2 AM-loaded and permeabilized cells, confocal imaging show mitochondrial gain of Ca2+ on release of SR Ca2+ by caffeine and a loss of mitochondrial Ca2+ on application of PF pulses. These signals are suppressed by Na+-free or CGP-37157- containing solutions, implicating mNCX in mediating the Ca2+ release process. We hypothesize that mitochondrial Ca2+ stores contribute significantly to normal and pathological cardiac Ca2+-signaling in response to mechanical stimuli, release of ROS and Ca2+- overload. Using freshly dissociated cardiomyocytes, we shall test this hypothesis using electrophysiological, confocal and TIRF fluorescence imaging and molecular techniques: Aim 1. To characterize the involvement of reactive oxygen species (ROS) in activating PF-triggered Ca2+ transients and explore if they are enhanced by conditions of Ca2+ loading. Aim 2. To characterize the PF-induced Ca2+ release with respect to the mitochondrial membrane potential (m), location and ionic mechanism. Aim 3. To characterize the molecular nature of the mechano-sensor and the signaling pathway that leads to mitochondrial Ca2+ releases. The proposed research investigates a novel Ca2+-signaling pathway that may be activated by the stresses and strains that occur in normally working atrial and ventricular myocardia and gains prominence when mitochondrial function is compromised. Since mitochondria play an important role in apoptosis, cardiomyopathy, ischemia-reperfusion injury, Leigh syndrome, and fatal infantile acidosis, it is likely that such a specific mitochondrial Ca2+ -signaling pathway plays a critical role in these pathologies. PUBLIC HEALTH RELEVANCE: Human heart has great energetic needs because of its large workload (equivalent to pushing 1 ton the length of a football field per day); where the required energy supply is provided by mitochondria occupying ~30% of the cellular volume. Since cardiac contractility in normal and abnormal heart is mediated by cycling of Ca2+ in the cell, it is likely that Ca2+ signaling in the heart may also be the mechanism by which energy supply from the mitochondria is regulated. The nature of the signals that transmit information between the generation of energy supply per beat from the mitochondria and the Ca2+ released from the SR to meet the contractile needs of the myocyte is the subject of the proposed research in both healthy and diseased hearts.

描述（由申请人提供）：该项目的长期目标是表征正常和异常心脏功能中Ca2+信号的多样性。这项研究集中在一种新型的线粒体Ca2+信号途径上，该途径是通过将心脏细胞暴露于“加压液”（PF）的生物学相关喷气机（PF）引发的。考虑到正常心跳的强度主要取决于SR的Ca2+诱导的Ca2+释放（CICR），PF触发的响应可能会增加CICR与线粒体能量产生的相互作用的良好控制和可塑性，这可能在各种压力中变得至关重要 - 与流有关的肌病。 我们最近的结果表明，PF脉冲激活相对较慢（〜1.5 s）的Ca2+瞬变，该瞬变来自一个小的细胞内Ca2+池，似乎与跨膜的进入或释放SR Ca2+，一氧化氮（NO）或IP3信号途径的释放无关。但取决于线粒体函数的完整性作为代谢偶联剂FCCP或线粒体Ca2+ Uniporter（MCU）阻止器，RU360抑制了线粒体函数。还原剂DTT和活性氧（ROS）清除型tempol以及线粒体NCX（MNCX）阻滞剂CGP-37157，也专门抑制了这些CA2+瞬变。在Rhod-2 AM负载和透化细胞中，共焦成像显示Ca2+的线粒体增益在咖啡因释放SR Ca2+时，咖啡因释放SR Ca2+，以及在施用PF脉冲时的线粒体Ca2+损失。这些信号被含有Na+ Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-Fre-FR-FR-FR-37157抑制，这意味着MNCX介导Ca2+释放过程。 我们假设线粒体Ca2+商店对机械刺激，ROS的释放和Ca2+过载的响应，对正常和病理心脏CA2+ - 信号显着贡献。使用新鲜分离的心肌细胞，我们应使用电生理，共聚焦和TIRF荧光成像和分子技术检验该假设：目的1。表征激活PF触发的Ca2+瞬变的反应性氧（ROS）的参与，并探索它们是否增强，并探索它们是否增强。 CA2+加载条件。 目的2。相对于线粒体膜电位（M），位置和离子机制，表征PF诱导的Ca2+释放。 目的3。表征机械传感器的分子性质和导致线粒体Ca2+释放的信号通路。 拟议的研究调查了一种新型的Ca2+信号途径，该途径可能会因正常工作心理和心室心肌的应力和菌株而激活，当线粒体功能受到损害时，可能会受到正常工作和心室心肌的突出。由于线粒体在凋亡，心肌病，缺血 - 再灌注损伤，Leigh综合征和致命的婴儿酸中毒中起重要作用，因此这种特定的线粒体Ca2+ signaling途径很可能在这些病理学中起关键作用。 公共卫生相关性：由于其大量工作量，人类心脏具有巨大的充满活力的需求（相当于每天的足球场长度）；线粒体占据了约30％的细胞体积的30％。由于正常和异常心脏的心脏收缩性是通过细胞中Ca2+循环循环介导的，因此心脏中的Ca2+信号传导也可能是调节线粒体能量供应的机制。在线粒体中每次节拍的产生和从SR释放的Ca2+之间传递信息的信号的性质，以满足肌细胞的收缩需求，这是拟议研究的主题。