Connexin Distribution in Physiological Versus Pathological Cardiac Hypertrophy

生理性与病理性心脏肥大中的连接蛋白分布

• 批准号: 7788252
• 负责人: GEORGE COOPER
• 金额: --
• 依托单位: RALPH H JOHNSON VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7788252
• 关键词: Accounting; Address; Adenoviruses; Adrenergic Agents; Adrenergic Agonists; Adrenergic Receptor; Adult; Affinity; Agonist; Aliquot; Animal Model; Animals; Applications Grants; Arrestins; Arrhythmia; Atrial Heart Septal Defects; Attenuated; Basic Science; Binding; Biopsy; Cadherins; Canis familiaris; Cardiac; Cardiac Myocytes; Caring; Catheterization; Cell Adhesion; Cell Nucleolus; Cell Nucleus; Cell surface; Cells; Characteristics; Chronic; Clinical; Complex; Confocal Microscopy; Congenital Heart Defects; Connexin 43; Connexins; Connexon; Control Animal; Cyclic AMP; Cytoplasm; Cytoskeleton; Data; Dependence; Diffuse; Diffusion; Distal; Docking; Down-Regulation; Dynein ATPase; Echocardiography; Electrocardiogram; Electrons; Elements; Environment; Event; Exhibits; Failure; Family; Family Felidae; Felis catus; Functional disorder; Funding; Gap Junctions; Genes; Genetic Transcription; Genetic screening method; Goals; Golgi Apparatus; Grant; Grant Review; Growth; Heart; Heart Diseases; Heart Hypertrophy; Heart failure; Homeostasis; Human; Hypertrophy; Immunoblotting; Infection; Interphase Cell; Intervention; Intracellular Transport; Kinesin; Lead; Left; Left ventricular structure; Life; Life Cycle Stages; Location; Lung; Lysosomes; MAP4; Maps; Measurement; Measures; Mechanics; Mediating; Medical center; Membrane; Membrane Transport Proteins; Messenger RNA; Metalcaptase; Methods; Microfilaments; Microtubule Depolymerization; Microtubule Proteins; Microtubule-Associated Proteins; Microtubules; Minor; Modeling; Molecular; Morphology; Motion; Motor; Mus; Myocardial; Myocardium; Myofibrils; Nocodazole; Normal Cell; Normal Range; Optics; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Phenotype; Phosphoric Monoester Hydrolases; Phosphotransferases; Physiological; Plus End of the Microtubule; Positioning Attribute; Prevention; Principal Investigator; Process; Program Reviews; Progress Reports; Property; Propranolol; Protein Biosynthesis; Protein Dephosphorylation; Protein Isoforms; Protein phosphatase; Proteins; Protocols documentation; Pulmonary artery structure; Recruitment Activity; Research; Research Support; Ribonucleoproteins; Ribosomal RNA; Ribosomes; Right ventricular structure; Right-On; Role; Sarcolemma; Science; Series; Site; Specificity; Staining method; Stains; Stimulus; Stress; Structure; System; Systolic Pressure; Telemetry; Testing; Therapeutic Intervention; Thinking; Time; Tissues; Transfection; Transgenic Mice; Translating; Translational Research; Translations; Transport Process; Transport Vesicles; Treatment Efficacy; Tubulin; Up-Regulation; Ventricular; Vesicle; Work; adrenergic; base; beta-adrenergic receptor; constriction; density; extracellular; gap junction channel; gene therapy; hemodynamics; improved; interest; macromolecule; member; messenger ribonucleoprotein; overexpression; p21-activated kinase 1; palliative; particle; pressure; prevent; programs; public health relevance; receptor; receptor internalization; receptor recycling; research study; response; small molecule; success; trans-Golgi Network

DESCRIPTION (provided by applicant): Research supported by this grant during the previous twenty-four years has been built around extensive data showing that cardiac structure, composition, and function each respond rapidly and reversibly to changes in hemodynamic load. The first set of studies supported by this grant used isolated cells, or cardiocytes, and intact animals to demonstrate the role of load as a central regulator of cardiocyte growth. The second set of studies supported by this grant, which also used load change as the primary experimental variable, led to our discovery of a dense cardiocyte microtubule network during severe pressure-overload cardiac hypertrophy that contrib- utes to the contractile dysfunction which occurs in this setting. The initial goals for the subsequent studies of this abnormal microtubule network were to determine how it contributes to the contractile dysfunction of hypertrophied myocardium. Major findings have been that 1) it is based both on increased tubulin, and thus microtubules, and on greater microtubule stability, 2) the major car- diac microtubule-stabilizing microtubule-associated protein, MAP4, is greatly upregulated in pressure overload hypertrophy and binds extensively to microtubules, and 3) contractile dysfunction is caused by viscous loading imposed on shortening myofilaments by the dense microtubule network. However, the most important normal role of the microtubules in an interphase cell such as the cardiocyte is not to determine cellular rheological properties but rather to subserve intracellular transport of macromolecules and vesicles via the microtubule-associated kinesin and dynein families of motor proteins. Indeed, this is an absolutely essential role in the extremely diffusion-restricted cytoplasm of the adult cardiocyte. For this reason, and because of the known inhibition of microtubule-dependent intracellular transport by excessive decoration of microtubules with MAPs, we next asked if microtubule-based transport of the activated 2-adrenergic receptor and/or mRNA - ribonucleoprotein complexes was inhibited by MAP4 binding to microtubules in pressure- overload hypertrophy. Such, in fact. was the case. Building on this most recent work, we propose to examine here the potential role of alterations in microtubule network organization and MAP4 binding in causing abnormal transport and localization of connexin43 [Cx43], a gap junction protein known to undergo functionally important alterations in quantity and localization during pathological cardiac hypertrophy. The basic research in the first objective will use isolated cells as well as oper- ated and transgenic mice to determine whether MAP4 decoration of microtubules, and the attendant densifica- tion of the microtubule network, inhibit the normal transport of Cx43 to gap junctions as well as Cx43-depen- dent electrophysiological function. The translational research in the second and third objectives will compare an equal degree & duration of pathological pressure vs. physiological volume overload hypertrophy. We will first extend the findings of the first objective to ask if MAP4 decoration of the dense microtubule network in pathological hypertrophy has a role in the altered Cx43 transport and localization that are important clinically in forming an arrhythmogenic substrate. We will then ask if 2-receptor blockade in pathological hypertrophy, which early data indicates will prevent the abnormal microtubule phenotype, will also prevent the abnormal Cx43 phenotype in this setting. In the first objective we will use murine models, and in the second and third objectives we will use our long- standing feline models of physiological versus pathological hypertrophy. While we recognize that it is prefer- able to use a single species, in this research the initial mechanistic portion can only be done in the mouse, but the later quantitative translational portions require very reproducible animal models that can be reliably and verifiably 2-blocked and have an equivalent degree and duration of physiological vs. pathological hypertrophy, with ex- tensively characterized cytoskeletal properties in each setting. PUBLIC HEALTH RELEVANCE: Pathological cardiac hypertrophy is one of the two most common serious cardiac abnormalities which we encounter in the care of our patients in VA Medical Centers. Our attempts to deal in a definitive as opposed to a palliative way with this problem must be based on a mechanistic understanding of the causes of this entity. Preliminary data for this grant application suggest that specific cytoskeletal changes accompanying severe pres- sure overload cardiac hypertrophy may not only be responsible for contractile dysfunction but may also be responsible, at least in part, for altered transport within the cardiac muscle cell that can lead to the rhythm disturbances that characterize heart failure. Since specific endogenous phosphatases and kinases regulate the molecular events that cause these cytoskeletal changes, this work has the potential to lead to very important therapeutic interventions.

描述（由申请人提供）： 在过去的24年里，这项资助支持的研究是建立在大量数据的基础上的，这些数据表明心脏的结构、组成和功能都对血流动力学负荷的变化做出了快速和可逆的反应。这项资助支持的第一组研究使用分离的细胞或心肌细胞和完整的动物来证明负荷作为心肌细胞生长的中央调节器的作用。由该基金支持的第二组研究也使用负荷变化作为主要实验变量，导致我们发现在严重压力超负荷心脏肥大期间密集的心脏细胞微管网络，这有助于在这种情况下发生的收缩功能障碍。 对这种异常微管网络的后续研究的最初目标是确定它如何导致肥厚心肌的收缩功能障碍。主要的发现是：1）它基于增加的微管蛋白和微管，以及更大的微管稳定性，2）主要的心脏微管稳定微管相关蛋白MAP 4在压力超负荷肥大中被极大地上调，并广泛地结合微管，和3）收缩功能障碍是由密集的微管网络施加在缩短的肌丝上的粘性负荷引起的。 然而，微管在间期细胞（如心肌细胞）中最重要的正常作用不是决定细胞的流变学性质，而是通过微管相关的驱动蛋白和动力蛋白家族的运动蛋白来辅助大分子和囊泡的细胞内转运。事实上，这是一个绝对必要的作用，在极端扩散限制的细胞质的成年心肌细胞。由于这个原因，并且由于已知通过用MAP过度修饰微管来抑制微管依赖性细胞内转运，我们接下来询问活化β 2-肾上腺素能受体和/或mRNA -核糖核蛋白复合物的基于微管的转运是否被压力超负荷肥大中与微管结合的MAP 4抑制。事实上，是这样的。就是这样。 基于这一最新的工作，我们建议在这里研究微管网络组织和MAP 4结合的改变在引起连接蛋白43 [Cx43]的异常转运和定位中的潜在作用，连接蛋白43是一种间隙连接蛋白，已知在病理性心脏肥大期间在数量和定位方面发生功能上重要的改变。第一个目标的基础研究将使用分离的细胞以及手术和转基因小鼠来确定微管的MAP 4修饰以及伴随的微管网络的致密化是否抑制Cx43向间隙连接的正常转运以及Cx43依赖的电生理功能。第二和第三个目标的转化研究将比较病理性压力与生理性容量超负荷肥大的同等程度和持续时间。我们将首先扩展第一个目标的发现，以询问病理性肥大中致密微管网络的MAP 4装饰是否在改变的Cx43转运和定位中起作用，这些转运和定位在临床上对形成致炎底物很重要。然后我们将询问，在病理性肥大中，早期数据表明2-受体阻断将防止异常微管表型，是否也将防止这种情况下的异常Cx43表型。 在第一个目标中，我们将使用鼠模型，在第二个和第三个目标中，我们将使用我们长期使用的生理性与病理性肥大的猫模型。虽然我们认识到优选使用单一物种，但在本研究中，最初的机制部分只能在小鼠中进行，但后来的定量翻译部分需要非常可重复的动物模型，这些动物模型可以可靠地和可验证地被2-阻断，并且具有生理性与病理性肥大的相等程度和持续时间，在每种情况下具有广泛表征的细胞骨架性质。 公共卫生关系： 病理性心脏肥大是我们在VA医疗中心护理患者时遇到的两种最常见的严重心脏异常之一。我们试图以决定性的而不是姑息性的方式处理这个问题，必须基于对这个实体的原因的机械理解。本基金申请的初步数据表明，伴随严重压力超负荷心脏肥大的特定细胞骨架变化可能不仅是收缩功能障碍的原因，而且可能至少部分是心肌细胞内转运改变的原因，这可能导致以心力衰竭为特征的节律紊乱。由于特定的内源性磷酸酶和激酶调节引起这些细胞骨架变化的分子事件，这项工作有可能导致非常重要的治疗干预。