Remodeling of the Transverse Tubular System and Ryanodine Receptor Clusters in Dy

Dy 中横管系统和 Ryanodine 受体簇的重塑

• 批准号: 8464196
• 负责人: Frank Bernd Sachse
• 金额: $ 37.42万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8464196
• 关键词: Adrenergic Receptor; Anterior; Architecture; Calcium; Canis familiaris; Cardiac; Cell Therapy; Cells; Confocal Microscopy; Contracts; Coupling; Data; Employee Strikes; Event; Failure; Heart failure; Image; Investigation; Laser Scanning Confocal Microscopy; Lateral; Left; Location; Measures; Microscopic; Modeling; Modification; Movement; Muscle Cells; Nature; Normal Cell; Phenotype; Probability; Process; Proteins; Relative (related person); Research Design; Resolution; Ryanodine Receptor Calcium Release Channel; Sarcolemma; Structure; Structure-Activity Relationship; System; Testing; Tissues; Tubular formation; Up-Regulation; Ventricular; Western Blotting; Work; base; cell type; density; heart cell; insight; lateral ventricle; novel; protein expression; protein structure; public health relevance; reconstruction; research study; restoration; two-dimensional

DESCRIPTION (provided by applicant): The applicants' long-term aim is to gain insights in structure-function relationships underlying excitation- contraction (EC) coupling in normal and diseased heart cells. In this study, the way that cellular architecture and calcium (Ca) handling proteins involved in EC coupling are modified in dyssynchronous heart failure (DHF) will be examined. Further studies will be used to establish to what extent these modifications can be reversed by cardiac resynchronization therapy (CRT). These investigations will be conducted on canine models of both DHF and CRT. Left ventricular cells from anterior and lateral locations will be studied because previous studies indicated a very different phenotype of those cells in DHF. In specific aim 1, three-dimensional reconstructions of the transverse tubular system (t-system) and associated ryanodine receptor (RyR) clusters will be obtained with scanning confocal microscopy. The discovery of non-junctional RyRs is a novel finding in mammalian ventricular cells. The extent to which the number of junctional RyR clusters exceeds non-junctional clusters will be established in control cells. In the proposed studies the extent and nature of structural and functional remodeling during DHF will be established. These studies are based on the hypothesis that reconstructed structures in control myocytes are significantly altered in cells from DHF ventricles. In particular, the hypothesis that the t-system is more reduced in lateral than anterior cells will be tested. The structure of t-tubules and RyRs in intact tissue will be compared with isolated cells. Preliminary data indicate that in cells from intact tissue the t-system is far more extensive than in isolated cells. The hypothesis that there remain significant numbers of non-junctional RyRs in cells in tissue will be tested. The extent to which DHF related changes in structure in intact tissue parallels the changes that were observed in isolated cells will also be investigated. In specific aim 2, the microscopic changes in Ca transients that produce contractions as a consequence of alterations in the t-system and proteins associated with EC coupling will be established. This includes measuring Ca movements in normal and DHF cells with extremely rapid two-dimensional confocal microscopy. We will test the hypothesis that Ca transients in DHF are significantly disorganized compared to controls. In addition spontaneous release events that have been suggested to be arrhythmogenic and reflect the distribution of junctional and non-junctions RyR clusters will be measured. In specific aim 3, we will test the hypothesis that CRT partially or completely reverses the effects of DHF on cellular architecture and Ca transients. Both, structural and functional alterations observed in DHF cells are expected to be partially or completely reversed in both cell types as a result of CRT. However, number and/or size of RyR clusters are not expected to be restored. Finally, we will investigate the hypothesis that the probability of spontaneous release events in CRT cells is increased due to up-regulation of adrenergic receptors.

描述（由申请人提供）：申请人的长期目的是在正常和患病的心脏细胞中获得兴奋 - 收缩（EC）耦合的结构功能关系的见解。在这项研究中，将检查参与EC偶联的蛋白质的细胞结构和钙（CA）处理方式，将在功能障碍心力衰竭（DHF）中修饰。进一步的研究将用于确定通过心脏重新同步治疗（CRT）可以逆转这些修饰的程度。这些研究将对DHF和CRT的犬类模型进行。将研究来自前部和横向位置的左心室细胞，因为先前的研究表明DHF中这些细胞的表型截然不同。在特定的目标1中，将使用扫描共聚焦显微镜获得横向管系统（T-System）和相关的ryanodine受体（RYR）簇的三维重建。在哺乳动物心室细胞中发现了非交连的Ryrs的发现。在对照细胞中将建立连接性RYR簇的数量超过非官方簇的程度。在拟议的研究中，将建立DHF期间结构和功能重塑的程度和性质。这些研究基于以下假设：对照肌细胞中的重建结构在DHF心室的细胞中显着改变。特别是，将测试侧向细胞中T系统更降低的假设。完整组织中T管和RYR的结构将与分离的细胞进行比较。初步数据表明，在完整组织的细胞中，T系统比孤立的细胞更广泛。将测试组织细胞中存在大量的非官方RYRS的假设。 DHF完整组织中结构的相关结构的相关程度与分离的细胞中观察到的变化也将被研究。在特定的目标2中，将建立由于T-System的变化和与EC耦合相关的蛋白质的变化而产生收缩的CA瞬变的显微镜变化。这包括测量正常和DHF细胞中的Ca运动，并具有极快的二维共聚焦显微镜。我们将检验以下假设：与对照组相比，DHF中的CA瞬变严重混乱。此外，将测量已提出心律不齐的自发释放事件，并反映了连接性和非障碍RYR簇的分布。在特定目标3中，我们将检验以下假设，即CRT部分或完全逆转DHF对细胞结构和CA瞬变的影响。在DHF细胞中观察到的结构和功能变化都被预期在CRT的两种细胞类型中都部分或完全逆转。但是，预计不会恢复RYR簇的数字和/或大小。最后，我们将研究以下假设：由于肾上腺素能受体的上调，CRT细胞中自发释放事件的可能性增加了。