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）： 在压力超负荷或缺血/再灌注等病理过程中，心肌细胞 (CM) 膜的脆性增加，可导致 CM 功能障碍、膜破裂并最终导致 CM 死亡。虽然这种现象已得到主要研究，但其背后的机制仍不完全明确。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 的附着部分是由整联蛋白复合物介导的。 Talin 位于 CM 内称为整合素的独特位点，可直接与 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肌节连接。Talin 是对整合素功能激活至关重要的关键衔接蛋白。初步数据表明，踝蛋白对于肋节的结构支撑至关重要，因此对于 CM 膜的稳定性至关重要。在 CM 中作为整合素-肌动蛋白连接体发挥作用，并且还调节整合素蛋白运输，因此它有助于维持肋结构并保持细胞的完整性。此外，当踝蛋白在心肌梗塞等损伤期间被钙蛋白酶裂解时，细胞脆弱、细胞破裂甚至细胞死亡都可能发生，这反过来又会导致细胞的细化。 ECM 产生和包括纤维化在内的有害重塑反应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。评估 talin 在心脏中的功能。首先，将评估导致 talin 缺乏的心脏心力衰竭的机制，重点是整合素运输如何参与这一过程的研究。将具体评估导致整合素内吞作用和降解增加的机制。了解这一过程很重要，因为它会影响 CM 与心脏内 ECM 的细胞粘附。研究也将定义这些细节。使用原子力显微镜进行的初步研究表明，talin 缺陷的肌细胞中的细胞张力发生变化，可导致细胞膜张力的降低。在 talin 缺乏的 CM 中，肋节的整合素更新增加，可能会导致细胞和整个心脏功能障碍。 钙蛋白酶对蛋白质的裂解与 CM 功能障碍有关。导致这样的假设：钙蛋白酶介导的踝蛋白裂解可导致肌膜破裂和细胞死亡，而废除这一过程可以保护心脏为了测试这一点，将在体内缺血再灌注的情况下使用抗钙蛋白酶的转基因，并且通常在这些模型中进行离体缺氧/再氧合模型。使用容易发生踝蛋白裂解的野生型小鼠和表达抗钙蛋白酶裂解的踝蛋白变体的转基因小鼠进行这些独特的模型以及源自它们的细胞将允许。直接测试预防踝蛋白裂解是否会保护 CM 和整个心脏免受应激相关损伤。最后，将进行详细研究，详细了解 CF 中的踝蛋白表达如何影响 ECM 的增殖、生长、形成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的一部分，即使心肌功能得以保留，也可能导致有害的心脏功能和心律失常。该项目的临床意义在于，大量 VA 患者存在各种原因的心力衰竭，需要经常住院治疗并重视门诊护理，明确心功能不全的根本原因。可能导致心力衰竭新疗法的研究至关重要，并将成为该提案的重点。