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）： 心肌细胞（CM）膜在压力超负荷或缺血/再灌注等病理过程中的脆性增加可导致CM功能障碍、膜破裂并最终导致CM死亡。虽然这一现象已被广泛研究，但其背后的机制仍不完全确定。保持CM膜的完整性需要CM与周围细胞外基质（ECM）的牢固且稳定的连接。CM与ECM的连接由整合素复合物介导，整合素复合物部分位于CM内称为costameres的独特位点。整联蛋白直接与ECM蛋白结合，但需要接头蛋白与细胞内的肌动蛋白细胞骨架和肌节连接。对于大多数细胞中整合素的功能活化也至关重要的关键适配器是Talin，这是该提议的第四个。初步数据表明，塔林是必不可少的costameres的结构支持，从而CM膜的稳定性。据推测，talin在CM中起着整合素-肌动蛋白连接体的作用，并且还调节整合素蛋白运输。因此，它有助于维持肋节结构并保持细胞的完整性。此外，当talin蛋白在损伤如心肌梗塞期间被钙蛋白酶切割时，可能发生细胞脆性、细胞破裂甚至细胞死亡。这反过来又可以导致详细说明 ECM产生和有害的重塑反应，包括纤维化。这种重塑大部分是由心脏成纤维细胞（CF）传播的，其中talin也高度表达，并且它可能在细胞生长中发挥重要作用。这项提案将进行一系列的研究，以评估talin在心脏中的功能。首先，将评估导致他林缺陷心脏心力衰竭的机制，重点是整合素转运如何参与这一过程。将专门评价导致整合素内吞作用和降解增加的机制研究。了解这一过程很重要，因为它可以影响CM与心脏内ECM的细胞粘附。这些细节以前都没有在CM中研究过。研究还将确定talin缺乏的肌细胞中细胞张力的改变如何导致costameric破坏。使用原子力显微镜的初步研究表明，从CM中删除talin降低了膜张力。研究将测试talin缺陷CM中的张力降低如何增加整合素在costameres的周转，可能产生较弱的细胞-ECM连接;导致细胞和整个心脏功能障碍。钙蛋白酶对蛋白质的切割与CM功能障碍有关。Talin蛋白被钙蛋白酶切割。这部分导致了钙蛋白酶介导的talin切割可以导致肌膜破裂和细胞死亡的假设，而取消这一过程可以保护心脏免受损害。为了测试这一点，钙蛋白酶抗性talin转基因将在体内缺血-再灌注和离体缺氧/再氧合模型的背景下使用。钙蛋白酶激活和talin切割通常发生在这些模型中。将使用易于发生talin裂解的野生型小鼠和表达talin变体（对钙蛋白酶裂解具有抗性）的转基因小鼠进行研究。这些独特的模型，以及从它们衍生的细胞，将允许直接测试，如果防止talin切割将保护CM和整个心脏免受压力相关的损伤。最后，将进行研究，详细说明talin在CF中的表达如何影响细胞外基质的增殖、生长、加工以及最终影响心脏的纤维化反应。这一点很重要，因为心脏纤维化是多种心脏病理的一部分，即使心肌功能得到保护，也可能导致有害的心脏功能和心律失常。提示talin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，需要频繁住院和关注门诊护理。确定心功能不全的根本原因，重要的是，可能导致心力衰竭新疗法的研究是必不可少的，并将成为本提案的重点。