Costamere Structure, Membrane Stability and Integrin Trafficking in the Normal and Diseased Heart

正常和患病心脏中的肋结构、膜稳定性和整合素运输

• 批准号: 9028289
• 负责人: Robert Scott Ross
• 金额: --
• 依托单位: VA SAN DIEGO HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9028289
• 关键词: Actins; Adaptor Signaling Protein; Affect; Ambulatory Care; Arrhythmia; Atomic Force Microscopy; Attention; Binding; Biological Models; Biological Preservation; Calpain; Cardiac; Cardiac Myocytes; Cell Adhesion; Cell Death; Cell Proliferation; Cell membrane; Cell model; Cells; Cellular biology; Cessation of life; Cleaved cell; Complex; Cre-LoxP; Cytoskeleton; Data; Diagnosis; Dilated Cardiomyopathy; Disease; Endocytosis; Environment; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Functional disorder; Growth; Health; Heart; Heart Diseases; Heart failure; Hospitalization; Hypertension; Hypoxia; Injury; Integrin Binding; Integrins; Ischemia; Lead; Link; Maintenance; Mediating; Membrane; Modeling; Molecular; Muscle; Muscle Cells; Myocardial; Myocardial Infarction; Myocardial dysfunction; Myocardium; Pathologic Processes; Pathology; Patients; Peptide Hydrolases; Plant Roots; Play; Prevention; Process; Production; Protein Isoforms; Proteins; Proteolysis; Reperfusion Therapy; Resistance; Role; Rupture; Sarcolemma; Sarcomeres; Series; Site; Stress; Structure; Supporting Cell; Switch Genes; System; Talin; Testing; Transgenes; Transgenic Model; Transgenic Organisms; Variant; Ventricular Remodeling; Wild Type Mouse; cell growth; cell type; clinically significant; coronary fibrosis; heart preservation; hemodynamics; improved; in vivo; mortality; mutant; novel therapeutics; palliative; pressure; protein expression; protein transport; public health relevance; response; trafficking

 DESCRIPTION (provided by applicant): Increased fragility of cardiac myocyte (CM) membranes during pathological processes such as pressure overload or ischemia/reperfusion, can lead to CM dysfunction, membrane rupture and ultimately CM death. While this phenomenon has been studied extensively, the mechanisms underlying it remain incompletely defined. Preservation of CM membrane integrity requires strong and stable connections of CMs with the surrounding extracellular matrix (ECM). CM attachment to the ECM is mediated by integrin complexes, which are in part localized at the unique site within CMs termed costameres. Integrins bind directly to ECM proteins but require adaptor proteins to link with the actin cytoskeleton and sarcomeres within the cell. A critical adapter that is also crucial for functional activation of integrins in most cells, is Talin, the fous of this proposal. Preliminary data show that Talin is essential for the structural support of costameres and thus for CM membrane stability. It is hypothesized that talin plays a role in CMs as an integrin-actin linker, and also regulates integrin protein trafficking. As such it contribute to maintenance of costamere structure and preserves the integrity of cells. Further, when talin protein is cleaved by calpain proteases during insults such as myocardial infarction, cellular fragility, cellular rupture and even cell death, can occur. This in turn can lead to elaboration of ECM production and deleterious remodeling responses which includes fibrosis. Much of this remodeling is propagated by cardiac fibroblasts (CF), where talin is also highly expressed and where it may play an important role in cell growth. This proposal will pursue a series of studies to assess the function of talin in the heart. First the mechanism(s) that lead to heart failure in he talin deficient heart will be evaluated with a focus on how integrin trafficking is involved in thi process. Study of the mechanisms that lead to increased integrin endocytosis and degradation will be specifically evaluated. Understanding this process is important since it can affect cell adhesion of CMs to the ECM within the heart. None of these details has been studied previously in CMs. Studies will also define how cell tension that is altered in talin deficient myocytes, can lead to costameric disruption. Preliminary studies using atomic force microscopy show that talin deletion from CMs reduces membrane tension. Studies will test how decreased tension in talin deficient CMs increases integrin turnover at the costameres, potentially producing weaker cell-ECM connections; leading to cellular and whole heart dysfunction. Cleavage of proteins by calpain proteases has been linked to CM dysfunction. Talin protein is cleaved by calpain. This in part lead to the hypothesis that calpain-mediated cleavage of talin can cause sarcolemmal rupture and cell death, and that abolition of this process can protect the heart from damage. To test this, a calpain-resistant talin transgene will be used in the context of in vivo ischemia-reperfusion, and ex vivo hypoxia/re-oxygenation models. Calpain activation and talin cleavage usually occurs in these models. Studies will be performed using wild-type mice prone to talin cleavage, and transgenic ones expressing the talin variant which is resistant to calpain cleavage. These unique models, and cells derived from them, will allow direct testing if prevention of talin cleavage will protect CMs and the whole heart from stress-related damage. Finally, studies will be pursued detailing how talin expression in CFs influences proliferation, growth, elaboration of ECM and ultimately, the fibrotic response of the heart. This is important since cardiac fibrosis occurs as part of multiple cardiac pathologies, and can lead to deleterious cardiac function and arrhythmias, even if myocardial function is preserved. It is suggested that talin may play important roles to modulate myocardial fibrosis. The clinical significance of this project is that heart failure of varied causes is found in a large number of VA patients, necessitating frequent hospitalizations and attention to outpatient care. Identification of root causes of cardiac dysfunction and importantly, studies which could lead to novel therapeutics for heart failure are essential, and will be the focus of this proposal.

 描述（由申请人提供）： 在诸如压力超负荷或缺血/再灌注之类的病理过程中，心肌细胞（CM）膜的脆弱性增加可导致CM功能障碍，膜破裂和最终导致CM死亡。尽管这种现象已经进行了广泛研究，但其基础机制仍未完全定义。 CM膜完整性的保留需要CM与周围细胞外基质（ECM）的稳定连接。 CM对ECM的附件是由整联蛋白复合物介导的，整联蛋白络合物部分位于CMS称为服装的独特地点。整联蛋白直接与ECM蛋白结合，但需要衔接蛋白与细胞内的肌动蛋白细胞骨架和肉瘤联系起来。对于大多数细胞中整联蛋白的功能激活也至关重要的关键适配器是塔林，这是该提案的影响。初步数据表明，塔林对于服装器的结构支持至关重要，因此对于CM膜稳定性至关重要。假设塔林在CMS作为整联蛋白 - 肌动蛋白接头起着作用，并且还调节整联蛋白蛋白运输。因此，它有助于维持Costamere结构并保留细胞的完整性。此外，可能会发生在侮辱性的心肌梗死，细胞脆弱，细胞破裂甚至细胞死亡时，钙蛋白酶蛋白被钙蛋白酶蛋白裂解时。反过来，这可能会导致 ECM生产和有害重塑反应，包括纤维化。这种重塑的大部分是由心脏成纤维细胞（CF）传播的，在该心脏成纤维细胞（CF）中，塔林也高度表达，并且可能在细胞生长中起重要作用。该建议将进行一系列研究，以评估塔林在心脏中的功能。首先，将评估导致He talin缺乏心脏心力衰竭的机制，重点是整合蛋白运输如何参与TH过程。将专门评估导致整联蛋白内吞和降解的机制的研究。了解此过程很重要，因为它会影响CMS对ECM内心内的细胞粘附。这些细节均未在CMS中研究。研究还将定义塔林缺乏肌细胞中改变的细胞张力如何导致顾客破坏。使用原子力显微镜进行初步研究表明，CMS的滑石缺失减少了膜张力。研究将测试塔林缺乏CMS的张力减少如何增加服装素的整联蛋白周转，从而可能产生较弱的细胞ECM连接；导致细胞和全心功能障碍。钙蛋白酶蛋白质裂解蛋白质与CM功能障碍有关。塔林蛋白被钙蛋白酶裂解。这部分导致了这样的假设，即calpain介导的塔林裂解会导致肌肉破裂和细胞死亡，并且废除该过程可以保护心脏免受损害。为了测试这一点，将使用耐钙蛋白酶的塔林变换，并在体内缺血再灌注和离体缺氧/重新氧化模型的背景下使用。钙蛋白酶激活和塔林裂解通常发生在这些模型中。研究将使用容易裂解的野生型小鼠进行研究，以及表达对钙蛋白酶裂解具有抗性的Talin变体的转基因。这些独特的模型以及从中得出的细胞将允许直接测试，如果预防塔林裂解将保护CMS和整个心脏免受与压力相关的损害。最后，将进行研究，详细介绍CFS中的Talin表达如何影响ECM的增殖，生长，阐述以及最终的心脏纤维化反应。这很重要，因为心脏纤维化是多种心脏病理的一部分，即使保留心肌功能也可以导致有害心脏功能和心律不齐。建议塔林可能在调节心肌纤维化中起重要作用。该项目的临床意义在于，在大量VA患者中发现了各种原因的心力衰竭，经常住院和对门诊护理的关注。鉴定心脏功能障碍的根本原因，重要的是，可能导致心力衰竭治疗的研究至关重要，这将是该建议的重点。